Abstract:

Stabilized compositions of specific binding agents to RANKL, specific
binding agents to TNF, and/or specific binding agents to IL-1R1 in
containers are provided. Methods of making and using such compositions
are also provided.

Claims:

2. The prefilled syringe of claim 1, wherein the specific binding agent is
selected from a specific binding agent to RANKL, a specific binding agent
to TNF, and a specific binding agent to IL-1R1.

3. The prefilled syringe of claim 2, wherein the specific binding agent is
selected from an antibody, a polyclonal antibody, a monoclonal antibody,
an antibody wherein the heavy chain and the light chain are connected by
a flexible linker, an Fv molecule, a maxibody, an antigen binding
fragment, a Fab fragment, a Fab' fragment, a F(ab')2 molecule, a
fully human antibody, a humanized antibody, and a chimeric antibody.

4. The prefilled syringe of claim 3, wherein the specific binding agent is
an antibody selected from an antibody that substantially inhibits binding
of RANKL to RANK, an antibody that substantially inhibits binding of TNF
to TNF-R, and an antibody that substantially inhibits binding of IL-1 to
IL-1R1.

5. The prefilled syringe of claim 4, wherein the antibody is an antibody
that substantially inhibits binding of RANKL to RANK, wherein the
antibody is αRANKL-1, wherein αRANKL-1 comprises a heavy
chain and a light chain, wherein the heavy chain comprises an amino acid
sequence as set forth in SEQ ID NO: 2 or a fragment thereof, and the
light chain comprises an amino acid sequence as set forth in SEQ ID NO: 4
or a fragment thereof.

6. The prefilled syringe of claim 4, wherein the antibody is an antibody
that substantially inhibits binding of IL-1 to IL-1R1.

7. The prefilled syringe of claim 1, wherein the composition further
comprises at least one additional pharmaceutical agent.

8. The prefilled syringe of claim 1, wherein the composition further
comprises at least one stabilizing agent and a buffering agent.

9. The prefilled syringe of claim 8, wherein at least one stabilizing
agent is a surfactant.

10. The prefilled syringe of claim 9, wherein the surfactant is selected
from polysorbate and polyoxypropylene-polyoxyethylene esters
(Pluronic®).

11. The prefilled syringe of claim 10, wherein the surfactant is
polysorbate.

12. The prefilled syringe of claim 11, wherein the polysorbate is selected
from polysorbate 20 and polysorbate 80.

13. The prefilled syringe of claim 9, wherein the surfactant is present at
a concentration of 0.001% to 1%.

14. The prefilled syringe of claim 13, wherein the surfactant is present
at a concentration of 0.002% to 0.5%.

15. The prefilled syringe of claim 14, wherein the surfactant is present
at a concentration of 0.004%.

16. The prefilled syringe of claim 14, wherein the surfactant is present
at a concentration of 0.01%.

17. The prefilled syringe of claim 8, wherein the pH of the composition is
below 6.6.

18. The prefilled syringe of claim 17, wherein the pH of the composition
is between 5.5 and 6.5.

19. The prefilled syringe of claim 18, wherein the pH of the composition
is 6.3.

20. The prefilled syringe of claim 17, wherein the pH of the composition
is between 4.5 and 5.5.

21. The prefilled syringe of claim 20, wherein the pH of the composition
is 5.2.

22. The prefilled syringe of claim 1, wherein the syringe comprises a
material comprising silicone, wherein the silicone is cross-linked.

23. The prefilled syringe of claim 1, wherein the syringe comprises a
material comprising silicone, wherein the silicone is baked.

28. A prefilled syringe containing a composition comprising a specific
binding agent, wherein a headspace between the composition and a syringe
closure is minimized, and wherein the specific binding agent contained in
the prefilled syringe is stabilized.

29. The prefilled syringe of claim 28, wherein the minimized headspace is
between 2.5 mm and 3.0 mm.

30. The prefilled syringe of claim 28, wherein the minimized headspace is
between 2.0 mm and 2.5 mm.

31. The prefilled syringe of claim 28, wherein the minimized headspace is
between 1.5 mm and 2.0 mm.

32. The prefilled syringe of claim 28, wherein the minimized headspace is
between 1.0 mm and 1.5 mm.

33. The prefilled syringe of claim 28, wherein the specific binding agent
is selected from a specific binding agent to RANKL, a specific binding
agent to TNF, and a specific binding agent to IL-1R1.

34. The prefilled syringe of claim 33, wherein the specific binding agent
is selected from an antibody, a polyclonal antibody, a monoclonal
antibody, an antibody wherein the heavy chain and the light chain are
connected by a flexible linker, an Fv molecule, a maxibody, an antigen
binding fragment, a Fab fragment, a Fab' fragment, a F(ab')2
molecule, a fully human antibody, a humanized antibody, and a chimeric
antibody.

35. The prefilled syringe of claim 34, wherein the specific binding agent
is an antibody selected from an antibody that substantially inhibits
binding of RANKL to RANK, an antibody that substantially inhibits binding
of TNF to TNF-R, and an antibody that substantially inhibits binding of
IL-1 to IL-1R1.

36. The prefilled syringe of claim 35, wherein the antibody is an antibody
that substantially inhibits binding of RANKL to RANK, wherein the
antibody is αRANKL-1, wherein αRANKL-1 comprises a heavy
chain and a light chain, wherein the heavy chain comprises an amino acid
sequence as set forth in SEQ ID NO: 2 or a fragment thereof, and the
light chain comprises an amino acid sequence as set forth in SEQ ID NO: 4
or a fragment thereof.

37. The prefilled syringe of claim 35, wherein the antibody is an antibody
that substantially inhibits binding of IL-1 to IL-1R1.

38. The prefilled syringe of claim 28, wherein the composition further
comprises at least one additional pharmaceutical agent.

39. The prefilled syringe of claim 28, wherein the composition further
comprises at least one stabilizing agent and a buffering agent.

40. The prefilled syringe of claim 39, wherein at least one stabilizing
agent is a surfactant.

41. The prefilled syringe of claim 40, wherein the surfactant is selected
from polysorbate and polyoxypropylene-polyoxyethylene esters
(Pluronic®).

42. The prefilled syringe of claim 41, wherein the surfactant is
polysorbate.

43. The prefilled syringe of claim 42, wherein the polysorbate is selected
from polysorbate 20 and polysorbate 80.

44. The prefilled syringe of claim 40, wherein the surfactant is present
at a concentration of 0.001% to 1%.

45. The prefilled syringe of claim 44, wherein the surfactant is present
at a concentration of 0.002% to 0.5%.

46. The prefilled syringe of claim 45, wherein the surfactant is present
at a concentration of 0.004%.

47. The prefilled syringe of claim 45, wherein the surfactant is present
at a concentration of 0.01%.

48. The prefilled syringe of claim 39, wherein the pH of the composition
is below 6.6.

49. The prefilled syringe of claim 48, wherein the pH of the composition
is between 5.5 and 6.5.

50. The prefilled syringe of claim 49, wherein the pH of the composition
is 6.3.

51. The prefilled syringe of claim 48, wherein the pH of the composition
is between 4.5 and 5.5.

52. The prefilled syringe of claim 51, wherein the pH of the composition
is 5.2.

53. The prefilled syringe of claim 28, wherein the syringe comprises a
material comprising silicone, wherein the silicone is cross-linked.

54. The prefilled syringe of claim 28, wherein the syringe comprises a
material comprising silicone, wherein the silicone is baked.

59. A method of preparing a prefilled syringe comprising introducing into
the syringe a composition comprising a specific binding agent such that a
headspace between the composition and a syringe closure is minimized, and
wherein the specific binding agent contained in the prefilled syringe is
stabilized.

60. The method of claim 59, wherein the specific binding agent is selected
from a specific binding agent to RANKL, a specific binding agent to TNF,
and a specific binding agent to IL-1R1.

61. The method of claim 60, wherein the specific binding agent is selected
from an antibody, a polyclonal antibody, a monoclonal antibody, an
antibody wherein the heavy chain and the light chain are connected by a
flexible linker, an Fv molecule, a maxibody, an antigen binding fragment,
a Fab fragment, a Fab' fragment, a F(ab')2 molecule, a fully human
antibody, a humanized antibody, and a chimeric antibody.

62. The method of claim 61, wherein the specific binding agent is an
antibody selected from an antibody that substantially inhibits binding of
RANKL to RANK, an antibody that substantially inhibits binding of TNF to
TNF-R, and an antibody that substantially inhibits binding of IL-1 to
IL-1R1.

63. The method of claim 62, wherein the antibody is an antibody that
substantially inhibits binding of RANKL to RANK, wherein the antibody is
αRANKL-1, wherein αRANKL-1 comprises a heavy chain and a
light chain, wherein the heavy chain comprises an amino acid sequence as
set forth in SEQ ID NO: 2 or a fragment thereof, and the light chain
comprises an amino acid sequence as set forth in SEQ ID NO: 4 or a
fragment thereof.

64. The method of claim 62, wherein the antibody is an antibody that
substantially inhibits binding of IL-1 to IL-1R1.

65. The method of claim 59, wherein the composition further comprises at
least one additional pharmaceutical agent.

66. The method of claim 59, wherein the composition further comprises at
least one stabilizing agent and a buffering agent.

67. The method of claim 66, wherein at least one stabilizing agent is a
surfactant.

68. The method of claim 67, wherein the surfactant is selected from
polysorbate and polyoxypropylene-polyoxyethylene esters (Pluronic®).

69. The method of claim 68, wherein the surfactant is polysorbate.

70. The method of claim 69, wherein the polysorbate is selected from
polysorbate 20 and polysorbate 80.

71. The method of claim 67, wherein the surfactant is present at a
concentration of 0.001% to 1%.

72. The method of claim 71, wherein the surfactant is present at a
concentration of 0.002% to 0.5%.

73. The method of claim 72, wherein the surfactant is present at a
concentration of 0.004%.

74. The method of claim 72, wherein the surfactant is present at a
concentration of 0.01%.

75. The method of claim 66, wherein the pH of the composition is below
6.6.

76. The method of claim 75, wherein the pH of the composition is between
5.5 and 6.5.

77. The method of claim 76, wherein the pH of the composition is 6.3.

78. The method of claim 75, wherein the pH of the composition is between
4.5 and 5.5.

79. The method of claim 78, wherein the pH of the composition is 5.2.

80. The method of claim 59, wherein the syringe comprises a material
comprising silicone, wherein the silicone is cross-linked.

81. The method of claim 59, wherein the syringe comprises a material
comprising silicone, wherein the silicone is baked.

86. The prefilled syringe of claim 1 or claim 28, wherein the specific
binding agent is at a concentration of 1 mg/ml to 150 mg/ml.

87. The method of claim 59, wherein the specific binding agent is at a
concentration of 1 mg/ml to 150 mg/ml.

88. A method for stabilizing a specific binding agent in a composition,
wherein the composition is contained in a prefilled syringe, comprising
placing the composition in the prefilled syringe such that a headspace
between the composition and a syringe closure is minimized, and wherein
the specific binding agent contained in the prefilled syringe is
stabilized.

Description:

[0001]This application claims the benefit of U.S. Provisional Application
No. 61/065,065, filed Feb. 7, 2008. U.S. Provisional Application No.
61/065,065 is incorporated herein by reference in its entirety for any
purpose.

FIELD

[0002]Stabilized compositions of specific binding agents to RANKL,
specific binding agents to TNF, and/or specific binding agents to IL-1R1
in containers are provided. Methods of making and using such compositions
are also provided.

BACKGROUND

[0003]Certain therapeutic compositions comprise specific binding agents.
In certain instances, therapeutic compositions are placed in containers,
for example, for storage and shipping. In certain instances, such
containers are compatible with storage and shipping conditions, as well
as the mode of administration, for example, including, but not limited
to, subcutaneous, intramuscular or intravenous injection. Certain
exemplary containers include, but are not limited to, an ampoule,
disposable syringe, including, but not limited to, disposable syringe
suitable for prefilling, and multiple dose vial made of glass or plastic.
In certain instances, a therapeutic composition is contained in a
prefilled syringe, for example, a syringe into which a manufacturer has
placed the therapeutic composition.

[0004]Therapeutic compositions in containers can, in certain instances,
form particles and/or show aggregation upon exposure to shipping and/or
storage conditions. Such compositions which exhibit particle formation
and/or aggregation, in certain instances, are not suitable for
administration and must be disposed of. It is desirable, in certain
instances, to provide stabilized therapeutic compositions in containers
which, when exposed to shipping and/or storage conditions, are less
susceptible to particle formation and/or aggregation.

[0006]In certain embodiments, a prefilled syringe containing a composition
comprising a specific binding agent is provided, wherein a headspace
between the composition and a syringe closure is minimized, and wherein
the specific binding agent contained in the prefilled syringe is
stabilized.

[0007]A method of preparing a prefilled syringe comprising introducing
into the syringe a composition comprising a specific binding agent such
that a headspace between the composition and a syringe closure is
minimized, and wherein the specific binding agent contained in the
prefilled syringe is stabilized.

[0008]In certain embodiments, a method for stabilizing a specific binding
agent in a composition is provided, wherein the composition is contained
in a prefilled syringe, comprising placing the composition in the
prefilled syringe such that a headspace between the composition and a
syringe closure is minimized, and wherein the specific binding agent
contained in the prefilled syringe is stabilized.

BRIEF DESCRIPTION OF THE FIGURES

[0009]FIG. 1 shows the stability of αRANKL-1 compositions at various
protein concentrations incubated in vials at 4° C. for 24 months,
and analyzed at various time points by native SEC-HPLC, according to the
work discussed in Example 1. (A) Percent main peak (monomer); (B) percent
aggregate (pre-peak).

[0010]FIG. 2 shows the stability under static conditions of αRANKL-1
compositions at various protein concentrations, after incubating in
prefilled glass luer lock syringes or prefilled glass staked needle
syringes at 4° C. for 24 weeks, and analyzed at various time
points by native SEC-HPLC, according to the work discussed in Example 1.

[0011]FIG. 3 shows the percent main peak (monomer) of αRANKL-1
compositions in a polysorbate-free formulation in COP plastic (Resin
CZ®) prefilled syringes after incubation at 4° C. for 4 weeks,
10 weeks, 22 weeks, 32 weeks, or 52 weeks, under static conditions or
after shipping, and analyzed at various times by native SEC-HPLC,
according to the work discussed in Example 1.

[0012]FIG. 4 shows the size distribution of sTNFR:Fc samples. The figure
shows the sub-visible particle size, as indicated by the intensity
weighted size distribution, of sTNFR:Fc samples subjected to various
prefilling and shipping conditions according to the work discussed in
Example 2.

[0013]FIG. 5 (A) is a schematic drawing of a staked-needle syringe and
syringe components; FIG. 5 (B) is a schematic drawing of a prefilled
syringe showing a headspace that is not minimized.

[0014]FIG. 6 shows a prefilled syringe containing a composition comprising
αRANKL-1 and a headspace, or a minimized headspace, according to
the work discussed in Example 2; (A) headspace not minimized, showing a
headspace of 4.5 mm; (B) minimized headspace showing (left side): a
minimized headspace of 1.5 mm with a meniscus; and (right side): a
minimized headspace with a visible air bubble.

[0016]FIG. 8 shows the amino acid sequence of the αRANKL-1 antibody
heavy chain (SEQ ID NO: 2). The IgG2 signal peptide is underlined, the
variable region is in capital letters and is not underlined, and the
constant region is in lower case.

[0017]FIG. 9 shows a cDNA sequence encoding the αRANKL-1 antibody
kappa light chain (SEQ ID NO: 3). The figure shows the DNA sequence of
the kappa chain expression plasmid sequence from an XbaI site through a
SalI site. The start codon begins at nucleotide 12; and the stop codon
begins at nucleotide 717.

[0018]FIG. 10 shows the amino acid sequence of the αRANKL-1 antibody
kappa light chain (SEQ ID NO: 4). The kappa signal peptide is underlined,
the variable region is in capital letters and not underlined, and the
constant region is in lower case.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0019]The section headings used herein are for organizational purposes
only and are not to be construed as limiting the subject matter
described. All documents or portions of documents cited in this
application, including but not limited to patents, patent applications,
articles, books, and treatises, are expressly incorporated by reference
herein in their entirety for any purpose. In the event that one or more
of the documents, or portions of documents, incorporated by reference
defines a term that contradicts that term's definition in this
application, this application controls.

[0020]Standard techniques may be used for recombinant DNA, oligonucleotide
synthesis, and tissue culture and transformation (e.g., electroporation,
lipofection). Enzymatic reactions and purification techniques may be
performed according to manufacturer's specifications or as commonly
accomplished in the art or as described herein. The foregoing techniques
and procedures may be generally performed according to conventional
methods well known in the art and as described in various general and
more specific references that are cited and discussed throughout the
present specification. See e.g., Sambrook et al. Molecular Cloning: A
Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y. (1989)). Unless specific definitions are provided,
the nomenclatures utilized in connection with, and the laboratory
procedures and techniques of, analytical chemistry, synthetic organic
chemistry, and medicinal and pharmaceutical chemistry described herein
are those well known and commonly used in the art. Standard techniques
may be used for chemical syntheses, chemical analyses, pharmaceutical
preparation, formulation, delivery, and treatment of patients.

[0021]In this application, the use of the singular includes the plural
unless specifically stated otherwise. In this application, the word "a"
or "an" means "at least one" unless specifically stated otherwise. In
this application, the use of "or" means "and/or" unless specifically
stated otherwise. In the context of a multiple dependent claim, the use
of "or" refers back to more than one preceding independent or dependent
claim in the alternative only. Furthermore, the use of the term
"including," as well as other forms, such as "includes" and "included,"
is not limiting. Also, terms such as "element" or "component" encompass
both elements or components comprising one unit and elements or
components that comprise more than one unit unless specifically stated
otherwise.

[0022]Receptor Activator of NF-κB Ligand (RANKL), also known as
Osteoprotegerin Ligand (OPGL), a member of the tumor necrosis factor
(TNF) family of cytokines, promotes formation of osteoclasts through
binding to the receptor, RANK. In certain instances, increased osteoclast
activity correlates with a number of osteopenic disorders, including, but
not limited to, post-menopausal osteoporosis, Paget's disease, lytic bone
metastases, and rheumatoid arthritis. Therefore, a reduction in RANKL
activity may result in a decrease in osteoclast activity and may reduce
the severity of osteopenic disorders. Certain specific binding agents to
RANKL, including, but not limited to, antibodies, have been described.
See, e.g., U.S. Publication No. 2004/0033535, published Feb. 19, 2004,
which is hereby incorporated by reference for any purpose.

[0023]Interleukin-1 (IL-1) is a cytokine associated with the inflammatory
response. In certain instances, IL-1 stimulates cellular responses by
interacting with a heterodimeric receptor complex comprised of two
transmembrane proteins, IL-1 receptor type I (IL-1R1) and IL-1 receptor
accessory protein (IL-1RAcP). It has been reported that IL-1 first binds
to IL-1R1; IL-1RAcP is then recruited to this complex (Greenfeder et al.,
1995, J. Biol. Chem. 270:13757-13765; Yoon and Dinarello, 1998, J.
Immunology 160:3170-3179; Cullinan et al., 1998, J. Immunology
161:5614-5620), followed by signal transduction resulting in the
induction of a cellular response. It has been postulated that, in certain
instances, preventing IL-1 signaling by inhibiting IL-1 from binding IL-1
receptor, for example, IL-1R1, may be useful therapeutically for treating
certain IL-1 mediated diseases. In certain instances, specific binding
agents to IL-1R1 inhibit IL-1 binding to IL-1 receptor. Certain specific
binding agents to IL-1R1, including, but not limited to, antibodies, have
been described. See, e.g., U.S. Publication No. 2004/0097712, published
May 20, 2004, which is hereby incorporated by reference for any purpose.

[0024]Tumor necrosis factor-α (TNFα, also known as cachectin)
and tumor necrosis factor-β (TNFβ, also known as lymphotoxin)
are homologous mammalian endogenous secretory proteins capable of
inducing a wide variety of effects on a large number of cell types. The
significant similarities in the structural and functional characteristics
of these two cytokines have resulted in their collective description as
"TNF." Complementary cDNA clones encoding TNFα (Pennica et al.,
Nature 312:724, 1984) and TNFβ (Gray et al., Nature 312:721, 1984)
have been isolated, permitting further structural and biological
characterization of TNF.

[0026]As utilized in accordance with the present disclosure, the following
terms, unless otherwise indicated, shall be understood to have the
following meanings.

[0027]The term "receptor activator of NF-κB ligand" or "RANKL"
refers to a polypeptide which promotes formation of osteoclasts through
binding to a receptor activator of NF-κB ("RANK"). RANKL is also
called "osteoprotegerin ligand" or "OPGL." The term "RANKL" includes
fragments of RANKL, as well as related polypeptides, which include, but
are not limited to, allelic variants, splice variants, derivative
variants, substitution variants, deletion variants, and/or insertion
variants, fusion polypeptides, and interspecies homologs. In certain
embodiments, a RANKL polypeptide includes terminal residues, such as, but
not limited to, leader sequence residues, targeting residues, amino
terminal methionine residues, lysine residues, tag residues and/or fusion
protein residues.

[0028]The term "interleukin-1 receptor type 1" or "IL-1R1" refers to a
polypeptide which is a transmembrane receptor that is stimulated by an
inflammatory cytokine known as interleukin-1 ("IL-1"). The term "IL-1R1"
includes fragments of IL-1R1, as well as related polypeptides, which
include, but are not limited to, allelic variants, splice variants,
derivative variants, substitution variants, deletion variants, and/or
insertion variants, fusion polypeptides, and interspecies homologs. In
certain embodiments, an IL-1R1 polypeptide includes terminal residues,
such as, but not limited to, leader sequence residues, targeting
residues, amino terminal methionine residues, lysine residues, tag
residues and/or fusion protein residues.

[0029]The term "TNF receptor" or "TNF-R" refers to a polypeptide having an
amino acid sequence of a native mammalian TNF receptor, or fragments
thereof, as well as related polypeptides, which include, but are not
limited to, allelic variants, splice variants, derivative variants,
substitution variants, deletion variants, and/or insertion variants,
fusion polypeptides, and interspecies homologs. Certain exemplary methods
and assays to determine biological activity of TNF-R have been described,
e.g., in U.S. Pat. No. 5,945,397; and Mohler et al., J. Immunol.
151:1548-1561 (1993). In certain embodiments, a TNF receptor includes
terminal residues, such as, but not limited to, leader sequence residues,
targeting residues, amino terminal methionine residues, lysine residues,
tag residues and/or fusion protein residues. TNF-R is capable of binding
TNF ligand. In certain embodiments, TNF-R transduces a biological signal
initiated by a TNF ligand binding to a cell. In certain embodiments,
TNF-R is capable of binding anti-TNF-R antibodies raised against TNF-R
from natural (i.e., nonrecombinant) sources. The mature full-length human
TNF-R is a glycoprotein having a molecular weight of about 80 kilodaltons
(kDa). The terms "TNF receptor" or "TNF-R" include, but are not limited
to, variants or subunits of native polypeptides having at least 20 amino
acids and which exhibit at least some biological activity in common with
TNF-R, for example, soluble TNF-R constructs which are devoid of a
transmembrane region (and are secreted from the cell) but retain the
ability to bind TNF. Various exemplary TNF-Rs, including soluble TNF-Rs,
are disclosed, for example, in U.S. Pat. No. 5,945,397 and Mohler et al.,
J. Immunol. 151:1548-1561 (1993). Native human TNF-R is disclosed, for
example, in U.S. Pat. No. 5,945,397, Smith et al., Science 248:1019-1023
(1990), Loetscher et al., Cell 61: 351-359 (1990), and Schall et al.,
Cell 61: 361-370 (1990).

[0030]The term "soluble TNF-R" or "sTNF-R" refers to a polypeptide having
an amino acid sequence corresponding to all or part of the extracellular
region of a native TNF-R, for example, including but not limited to,
huTNF-RΔ235, huTNF-RΔ185 and huTNF-RΔ163, or a variant
of amino acids 1-163, amino acids 1-185, or amino acids 1-235 of native
human TNF-R as described in Smith et al., Science 248:1019-1023 (1990),
and which are biologically active in that they bind to TNF ligand. In
certain embodiments, sTNF-R is etanercept. Etanercept is a recombinant
fusion protein that contains the extracellular domain of the p75 sTNF-R
attached to a Fc fragment of a human IgG antibody. The amino acid
sequence of etanercept was published in Clinical Pharmacology and
Therapeutics 66(2):209, 1999, incorporated herein by reference, and the
protein is available for sale under the tradename Enbrel® (Amgen
Inc.).

[0031]The nomenclature for TNF-R and sTNF-R follows the convention of
naming the protein (e.g., TNF-R) preceded by either hu (for human) or mu
(for murine) and followed by a Δ (to designate a deletion) and the
number of the C-terminal amino acid. For example, huTNF-RΔ235
refers to human TNF-R having Asp235 as the C-terminal amino acid
(i.e., a polypeptide having the sequence of amino acids 1-235 of native
human TNF-R as described in Smith et al., Science 248:1019-1023 (1990)).
In the absence of any human or murine species designation, TNF-R refers
generically to mammalian TNF-R. Similarly, in the absence of any specific
designation for deletion mutants, the term TNF-R means all forms of
TNF-R, including variants which possess TNF-R biological activity.
Certain exemplary TNF-Rs include polypeptides which vary from the
sequences described above by one or more substitutions, deletions, or
additions, and which retain the ability to bind TNF or inhibit TNF signal
transduction activity via cell surface bound TNF receptor proteins, for
example huTNF-RΔx, wherein x is selected from any one of amino
acids 163-235 of native human TNF-R as described in Smith et al., Science
248:1019-1023 (1990). In certain embodiments, analogous deletions are
made to murine TNF-R ("muTNF-R"). In certain embodiments, inhibition of
TNF signal transduction activity is determined by transfecting cells with
recombinant TNF-R DNAs to obtain recombinant receptor expression. In such
embodiments, the transfected cells are then contacted with TNF and the
resulting metabolic effects examined. If an effect results which is
attributable to the action of the ligand, then the recombinant receptor
has signal transduction activity. Certain exemplary procedures for
determining whether a polypeptide has signal transduction activity are
disclosed, e.g., in Idzerda et al., J. Exp. Med. 171:861 (1990); Curtis
et al., Proc. Natl. Acad. Sci. USA 86:3045 (1989); Prywes et al., EMBO J.
5:2179 (1986) and Chou et al., J. Biol. Chem. 262:1842 (1987).
Alternatively, in certain embodiments, primary cells or cell lines which
express an endogenous TNF receptor and have a detectable biological
response to TNF are utilized.

[0032]The term "comprising," when used in connection with an amino acid
sequence, means that a compound may include additional amino acids on
either or both of the N- or C-termini of the given sequence.

[0033]In the context of polypeptides, two or more polypeptides are
"operably linked" if each linked polypeptide is able to function in its
intended manner. A polypeptide that is able to function in its intended
manner when operably linked to another polypeptide may or may not be able
to function in its intended manner when not operably linked to another
polypeptide. For example, in certain embodiments, a first polypeptide may
be unable to function in its intended manner when unlinked, but may be
stabilized by being linked to a second polypeptide such that it becomes
able to function in its intended manner. Alternatively, in certain
embodiments, a first polypeptide may be able to function in its intended
manner when unlinked, and may retain that ability when operably linked to
a second polypeptide.

[0034]As used herein, two or more polypeptides are "fused" when the two or
more polypeptides are linked by translating them as a single contiguous
polypeptide sequence or by synthesizing them as a single contiguous
polypeptide sequence. In certain embodiments, two or more fused
polypeptides may have been translated in vivo from two or more operably
linked polynucleotide coding sequences. In certain embodiments, two or
more fused polypeptides may have been translated in vitro from two or
more operably linked polynucleotide coding sequences.

[0035]As used herein, two or more polypeptides are "operably fused" if
each linked polypeptide is able to function in its intended manner.

[0036]In certain embodiments, a first polypeptide that contains two or
more distinct polypeptide units is considered to be linked to a second
polypeptide so long as at least one of the distinct polypeptide units of
the first polypeptide is linked to the second polypeptide.

[0037]In certain embodiments, the language "a first polypeptide linked to
a second polypeptide" encompasses situations where: (a) only one molecule
of a first polypeptide is linked to only one molecule of a second
polypeptide; (b) only one molecule of a first polypeptide is linked to
more than one molecule of a second polypeptide; (c) more than one
molecule of a first polypeptide is linked to only one molecule of a
second polypeptide; and (d) more than one molecule of a first polypeptide
is linked to more than one molecule of a second polypeptide. In certain
embodiments, when a linked molecule comprises more than one molecule of a
first polypeptide and only one molecule of a second polypeptide, all or
fewer than all of the molecules of the first polypeptide may be
covalently or noncovalently linked to the second polypeptide. In certain
embodiments, when a linked molecule comprises more than one molecule of a
first polypeptide, one or more molecules of the first polypeptide may be
covalently or noncovalently linked to other molecules of the first
polypeptide.

[0038]As used herein, a "flexible linker" refers to any linker that is not
predicted by one skilled in the art, according to its chemical structure,
to be fixed in three-dimensional space. In certain embodiments, a peptide
linker comprising three or more amino acids is a flexible linker.

[0039]In the context of polypeptides, two or more polypeptides are
"attached" if a first polypeptide is fused, operably fused, linked,
and/or operably linked to one or more polypeptides.

[0040]The term "specific binding agent" refers to a natural or non-natural
molecule that specifically binds to a target. Examples of specific
binding agents include, but are not limited to, proteins, peptides,
nucleic acids, carbohydrates, lipids; and small molecule compounds. In
certain embodiments, a specific binding agent is an immunoglobulin. In
certain embodiments, a specific binding agent is an immunoglobulin
fragment. In certain embodiments, a specific binding agent is an
antibody. In certain embodiments, a specific binding agent is an antigen
binding region.

[0041]The term "specific binding agent to RANKL" refers to a specific
binding agent that specifically binds any portion of RANKL. In certain
embodiments, a specific binding agent to RANKL is an immunoglobulin. In
certain embodiments, a specific binding agent to RANKL is an
immunoglobulin fragment. In certain embodiments, a specific binding agent
to RANKL is an antibody to RANKL. In certain embodiments, a specific
binding agent is an antigen binding region.

[0042]The term "specific binding agent to IL-1R1" refers to a specific
binding agent that specifically binds any portion of IL-1R1. In certain
embodiments, a specific binding agent to IL-1R1 is an immunoglobulin. In
certain embodiments, a specific binding agent to IL-1R1 is an
immunoglobulin fragment. In certain embodiments, a specific binding agent
to IL-1R1 is an antibody to IL-1R1. In certain embodiments, a specific
binding agent is an antigen binding region.

[0043]The term "specific binding agent to TNF" refers to a specific
binding agent that specifically binds any portion of TNF. In certain
embodiments, a specific binding agent to TNF is a polypeptide. In certain
embodiments, a specific binding agent to TNF is a soluble polypeptide. In
certain embodiments, a specific binding agent to TNF is a soluble
polypeptide operably fused to a second polypeptide, wherein the second
polypeptide is not a specific binding agent to TNF. Such second
polypeptides include for example, but are not limited to, Fc and Fc
fragment. In certain embodiments, a specific binding agent to TNF is an
immunoglobulin. In certain embodiments, a specific binding agent to TNF
is an immunoglobulin fragment. In certain embodiments, a specific binding
agent to TNF is an antibody to TNF. In certain embodiments, a specific
binding agent is an antigen binding region.

[0044]The term "specific binding agent to TNF-R" refers to a specific
binding agent that specifically binds any portion of TNF-R. In certain
embodiments, a specific binding agent to TNF-R is an immunoglobulin. In
certain embodiments, a specific binding agent to TNF-R is an
immunoglobulin fragment. In certain embodiments, a specific binding agent
to TNF-R is an antibody to TNF-R. In certain embodiments, a specific
binding agent is an antigen binding region.

[0045]The term "specifically binds" refers to the ability of a specific
binding agent to bind to a target with greater affinity than it binds to
a non-target. In certain embodiments, specific binding refers to binding
for a target with an affinity that is at least 10, 50, 100, 250, 500, or
1000 times greater than the affinity for a non-target. In certain
embodiments, affinity is determined by an affinity ELISA assay. In
certain embodiments, affinity is determined by a BIAcore® assay. In
certain embodiments, affinity is determined by a kinetic method. In
certain embodiments, affinity is determined by an equilibrium/solution
method.

[0046]The term "target" refers to a molecule or a portion of a molecule
capable of being bound by a specific binding agent. In certain
embodiments, a target may have one or more epitopes. In certain
embodiments, a target is an antigen.

[0047]The term "epitope" refers to a portion of a molecule capable of
being bound by a specific binding agent. Exemplary epitopes may comprise
any polypeptide determinant capable of specific binding to an
immunoglobulin and/or T-cell receptor. Exemplary epitope determinants
include, but are not limited to, chemically active surface groupings of
molecules, for example, but not limited to, amino acids, sugar side
chains, phosphoryl groups, and sulfonyl groups. In certain embodiments,
epitope determinants may have specific three dimensional structural
characteristics, and/or specific charge characteristics. In certain
embodiments, an epitope is a region of an antigen that is bound by an
antibody. Epitopes may be contiguous or non-contiguous. In certain
embodiments, epitopes may be mimetic in that they comprise a three
dimensional structure that is similar to an epitope used to generate the
antibody, yet comprise none or only some of the amino acid residues found
in that epitope used to generate the antibody.

[0048]"Antibody" or "antibody peptide(s)" both refer to an intact
antibody, or a fragment thereof. In certain embodiments, the fragment
includes contiguous portions of an intact antibody. In certain
embodiments, the fragment includes non-contiguous portions of an intact
antibody. In certain embodiments, the antibody fragment may be a binding
fragment that competes with the intact antibody for specific binding. The
term "antibody" also encompasses polyclonal antibodies and monoclonal
antibodies. In certain embodiments, binding fragments are produced by
recombinant DNA techniques. In certain embodiments, binding fragments are
produced by enzymatic or chemical cleavage of intact antibodies. Binding
fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv,
maxibodies, and single-chain antibodies. Non-antigen binding fragments
include, but are not limited to, Fc fragments.

[0049]The term "polyclonal antibody" refers to a heterogeneous mixture of
antibodies that bind to different epitopes of the same antigen.

[0050]The term "monoclonal antibodies" refers to a collection of
antibodies encoded by the same nucleic acid molecule. In certain
embodiments, monoclonal antibodies are produced by a single hybridoma or
other cell line, or by a transgenic mammal. Monoclonal antibodies
typically recognize the same epitope. The term "monoclonal" is not
limited to any particular method for making an antibody.

[0051]"Chimeric antibody" refers to an antibody that has an antibody
variable region of a first species fused to another molecule, for
example, an antibody constant region of another second species. See,
e.g., U.S. Pat. No. 4,816,567 and Morrison et al., Proc Natl Acad Sci
(USA), 81:6851-6855 (1985). In certain embodiments, the first species may
be different from the second species. In certain embodiments, the first
species may be the same as the second species. In certain embodiments, a
chimeric antibody is a CDR-grafted antibody.

[0052]The term "CDR-grafted antibody" refers to an antibody in which the
CDR from one antibody is inserted into the framework of another antibody.
In certain embodiments, the antibody from which the CDR is derived and
the antibody from which the framework is derived are of different
species. In certain embodiments, the antibody from which the CDR is
derived and the antibody from which the framework is derived are of
different isotypes.

[0053]The term "multi-specific antibody" refers to an antibody wherein two
or more variable regions bind to different epitopes. The epitopes may be
on the same or different targets. In certain embodiments, a
multi-specific antibody is a "bi-specific antibody," which recognizes two
different epitopes on the same or different antigens.

[0054]The term "catalytic antibody" refers to an antibody in which one or
more catalytic moieties is attached. In certain embodiments, a catalytic
antibody is a cytotoxic antibody, which comprise a cytotoxic moiety.

[0055]The term "humanized antibody" refers to an antibody in which all or
part of an antibody framework region is derived from a human, but all or
part of one or more CDR regions is derived from another species, for
example, including, but not limited to, a mouse.

[0056]The term "fully human antibody" refers to an antibody in which both
the CDR and the framework comprise substantially human sequences. In
certain embodiments, fully human antibodies are produced in non-human
mammals, including, but not limited to, mice, rats, and lagomorphs. In
certain embodiments, fully human antibodies are produced in hybridoma
cells. In certain embodiments, fully human antibodies are produced
recombinantly.

[0057]The term "heavy chain" includes any polypeptide having sufficient
variable region sequence to confer specificity for a target. A
full-length heavy chain includes a variable region domain, VH, and
three constant region domains, CH1, CH2, and CH3. The
VH domain is at the amino-terminus of the polypeptide, and the
CH3 domain is at the carboxy-terminus. The term "heavy chain", as
used herein, encompasses a full-length heavy chain and fragments thereof.

[0058]The term "light chain" includes any polypeptide having sufficient
variable region sequence to confer specificity for a target. A
full-length light chain includes a variable region domain, VL, and a
constant region domain, CL. Like the heavy chain, the variable
region domain of the light chain is at the amino-terminus of the
polypeptide. The term "light chain", as used herein, encompasses a
full-length light chain and fragments thereof.

[0059]The term "Fab fragment" refers to an antibody comprising one light
chain and the CH1 and variable regions of one heavy chain. The heavy
chain of a Fab fragment cannot form a disulfide bond with another heavy
chain. In certain embodiments, the heavy chain of a Fab fragment forms a
disulfide bond with the light chain of a Fab fragment.

[0060]The term "Fab' fragment" refers to an antibody comprising one light
chain, the variable and CH1 regions of one heavy chain, and some of
the constant region between the CH1 and CH2 domains of the
heavy chain. In certain embodiments, an interchain disulfide bond can be
formed between two heavy chains of an Fab' fragment to form a
F(ab')2 molecule.

[0061]The term "F(ab')2 molecule" refers to an antibody comprising
two Fab' fragments connected by an interchain disulfide bond formed
between two heavy chains.

[0062]An "Fv molecule" comprises the variable regions from both the heavy
and light chains, but lacks the constant regions. A single chain variable
fragment (scFv) comprises variable regions from both a heavy and a light
chain wherein the heavy and light chain variable regions are fused to
form a single polypeptide chain which forms an antigen-binding region. In
certain embodiments, a scFV comprises a single polypeptide chain. A
single-chain antibody comprises a scFV. In certain embodiments, a
single-chain antibody comprises one or more additional polypeptides fused
to a scFv. Exemplary additional polypeptides include, but are not limited
to, one or more constant regions. Exemplary single-chain antibodies are
discussed, e.g., in WO 88/01649 and U.S. Pat. Nos. 4,946,778 and
5,260,203.

[0063]The term "maxibody" refers to a scFv fused (may be by a linker or
direct attachment) to an Fc or an Fc fragment. In certain embodiments, a
single chain antibody is a maxibody. In certain embodiments, a single
chain antibody is a maxibody that binds to HGF. Exemplary Ig-like
domain-Fc fusions are disclosed in U.S. Pat. No. 6,117,655.

[0064]An "Fc fragment" comprises the CH2 and CH3 domains of the
heavy chain and contains some of the constant region, between the
CH1 and CH2 domains, such that an interchain disulfide bond can
be formed between two heavy chains.

[0065]The terms "variable region" and "variable domain" are used
interchangeably herein to refer to a portion of the light and/or heavy
chains of an antibody. In various instances, variable domains include
approximately the amino-terminal 120 to 130 amino acids in the heavy
chain and about 100 to 110 amino-terminal amino acids in the light chain.
In certain embodiments, variable regions of different antibodies differ
extensively in amino acid sequence even among antibodies of the same
species. The variable region of an antibody, in various instances,
determines specificity of a particular antibody for its target.

[0066]The term "antigen binding fragment" refers to a polypeptide fragment
comprising at least the variable domain of an immunoglobulin heavy chain
and at least the variable domain of an immunoglobulin light chain. In
certain embodiments, an antigen binding fragment is capable of binding to
a ligand, preventing binding of the ligand to its receptor, and thereby
interrupting a biological response resulting from ligand binding to the
receptor. In certain embodiments, an antigen binding fragment is capable
of binding to a receptor, preventing binding of the ligand to its
receptor, and thereby interrupting a biological response resulting from
ligand binding to the receptor. In certain embodiments, an antigen
binding fragment is capable of binding a receptor and activating that
receptor. In certain embodiments, an antigen binding fragment is capable
of binding a receptor and inactivating that receptor.

[0067]The term "naturally-occurring" as applied to an object refers to the
fact that an object can be found in nature. For example, a polypeptide or
polynucleotide sequence that is present in an organism (including
viruses) that can be isolated from a source in nature and which has not
been intentionally modified by man in the laboratory or otherwise is
naturally-occurring.

[0068]The term "isolated polynucleotide" refers to a polynucleotide of
genomic, cDNA, or synthetic origin or some combination thereof, which by
virtue of its origin the "isolated polynucleotide" (1) is not associated
with all or a portion of a polynucleotide in which the "isolated
polynucleotide" is found in nature, (2) is linked to a polynucleotide
which it is not linked to in nature, or (3) does not occur in nature as
part of a larger sequence.

[0069]The term "operably linked" refers to components that are in a
relationship permitting them to function in their intended manner. For
example, in the context of a polynucleotide sequence, a control sequence
may be "operably linked" to a coding sequence when the control sequence
and coding sequence are in association with each other in such a way that
expression of the coding sequence is achieved under conditions compatible
with the functioning of the control sequence.

[0070]The term "control sequence" refers to polynucleotide sequences which
may effect the expression and processing of coding sequences with which
they are in association. The nature of such control sequences may differ
depending upon the host organism. Certain exemplary control sequences for
prokaryotes include, but are not limited to, promoters, ribosomal binding
sites, and transcription termination sequences. Certain exemplary control
sequences for eukaryotes include, but are not limited to, promoters,
enhancers, and transcription termination sequences. In certain
embodiments, "control sequences" can include leader sequences and/or
fusion partner sequences.

[0071]The terms "isolated polypeptide" and "isolated peptide" refer to any
polypeptide that (1) is free of at least some proteins with which it
would normally be found, (2) is essentially free of other proteins from
the same source, e.g., from the same species, (3) is expressed by a cell
from a different species, or (4) does not occur in nature.

[0072]The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein and refer to a polymer of two or more amino acids
joined to each other by peptide bonds or modified peptide bonds, i.e.,
peptide isosteres. The terms apply to amino acid polymers containing
naturally occurring amino acids as well as amino acid polymers in which
one or more amino acid residues is a non-naturally occurring amino acid
or a chemical analogue of a naturally occurring amino acid. An amino acid
polymer may contain one or more amino acid residues that has been
modified by one or more natural processes, such as post-translational
processing, and/or one or more amino acid residues that has been modified
by one or more chemical modification techniques known in the art.

[0073]As used herein, the twenty conventional amino acids and their
abbreviations follow conventional usage. See Immunology--A Synthesis (2nd
Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,
Sunderland, Mass. (1991)). In certain embodiments, one or more
unconventional amino acids may be incorporated into a polypeptide. The
term "unconventional amino acid" refers to any amino acid that is not one
of the twenty conventional amino acids. The term "non-naturally occurring
amino acids" refers to amino acids that are not found in nature.
Non-naturally occurring amino acids are a subset of unconventional amino
acids. Unconventional amino acids include, but are not limited to,
stereoisomers (e.g., D-amino acids) of the twenty conventional amino
acids, unnatural amino acids such as α-,α-disubstituted amino
acids, N-alkyl amino acids, lactic acid, homoserine, homocysteine,
4-hydroxyproline, γ-carboxyglutamate,
ε-N,N,N-trimethyllysine, ε-N-acetyllysine,
O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,
5-hydroxylysine, σ-N-methylarginine, and other similar amino acids
and imino acids (e.g., 4-hydroxyproline) known in the art. In the
polypeptide notation used herein, the left-hand direction is the amino
terminal direction and the right-hand direction is the carboxy-terminal
direction, in accordance with standard usage and convention.

[0074]A "fragment" of a reference polypeptide refers to a contiguous
stretch of amino acids from any portion of the reference polypeptide. A
fragment may be of any length that is less than the length of the
reference polypeptide.

[0075]A "variant" of a reference polypeptide refers to a polypeptide
having one or more amino acid substitutions, deletions, or insertions
relative to the reference polypeptide. In certain embodiments, a variant
of a reference polypeptide has an altered post-translational modification
site (i.e., a glycosylation site). In certain embodiments, a variant of a
reference polypeptide has altered disulfide connectivity. In certain
embodiments, both a reference polypeptide and a variant of a reference
polypeptide are specific binding agents. In certain embodiments, both a
reference polypeptide and a variant of a reference polypeptide are
antibodies.

[0076]Variants of a reference polypeptide include, but are not limited to,
glycosylation variants. Glycosylation variants include variants in which
the number and/or type of glycosylation sites have been altered as
compared to the reference polypeptide. In certain embodiments,
glycosylation variants of a reference polypeptide comprise a greater or a
lesser number of N-linked glycosylation sites than the reference
polypeptide. In certain embodiments, an N-linked glycosylation site is
characterized by the sequence Asn-X-Ser or Asn-X-Thr, wherein the amino
acid residue designated as X may be any amino acid residue except
proline. In certain embodiments, glycosylation variants of a reference
polypeptide comprise a rearrangement of N-linked carbohydrate chains
wherein one or more N-linked glycosylation sites (typically those that
are naturally occurring) are eliminated and one or more new N-linked
sites are created.

[0077]Variants of a reference polypeptide include, but are not limited to,
cysteine variants. In certain embodiments, cysteine variants include
variants in which one or more cysteine residues of the reference
polypeptide are replaced by one or more non-cysteine residues; and/or one
or more non-cysteine residues of the reference polypeptide are replaced
by one or more cysteine residues. Cysteine variants may be useful, in
certain embodiments, when a particular polypeptide must be refolded into
a biologically active conformation, e.g., after the isolation of
insoluble inclusion bodies. In certain embodiments, cysteine variants of
a reference polypeptide have fewer cysteine residues than the reference
polypeptide. In certain embodiments, cysteine variants of a reference
polypeptide have an even number of cysteines to minimize interactions
resulting from unpaired cysteines. In certain embodiments, cysteine
variants have more cysteine residues than the native protein.

[0078]In certain embodiments, conservative modifications to the heavy and
light chains of a particular antibody (and corresponding modifications to
the encoding nucleotides) will produce antibodies having functional and
chemical characteristics similar to those of the original antibody. In
contrast, in certain embodiments, substantial modifications in the
functional and/or chemical characteristics of a particular antibody may
be accomplished by selecting substitutions in the amino acid sequence of
the heavy and light chains that differ significantly in their effect on
maintaining (a) the structure of the molecular backbone in the area of
the substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or (c)
the bulk of the side chain.

[0079]Certain desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at the
time such substitutions are desired. In certain embodiments, amino acid
substitutions can be used to identify important residues of particular
antibodies, such as those which may increase or decrease the affinity of
the antibodies or the effector function of the antibodies.

[0080]In certain embodiments, the effects of an antibody may be evaluated
by measuring a reduction in the amount of symptoms of the disease. In
certain embodiments, the disease of interest may be caused by a pathogen.
In certain embodiments, a disease may be established in an animal host by
other methods including introduction of a substance (such as a
carcinogen) and genetic manipulation. In certain embodiments, effects may
be evaluated by detecting one or more adverse events in the animal host.
The term "adverse event" includes, but is not limited to, an adverse
reaction in an animal host that receives an antibody that is not present
in an animal host that does not receive the antibody. In certain
embodiments, adverse events include, but are not limited to, a fever, an
immune response to an antibody, inflammation, and/or death of the animal
host.

[0081]Various antibodies specific to an antigen may be produced in a
number of ways. In certain embodiments, an antigen containing an epitope
of interest may be introduced into an animal host (e.g., a mouse), thus
producing antibodies specific to that epitope. In certain instances,
antibodies specific to an epitope of interest may be obtained from
biological samples taken from hosts that were naturally exposed to the
epitope. In certain instances, introduction of human immunoglobulin (Ig)
loci into mice in which the endogenous Ig genes have been inactivated
offers the opportunity to obtain human monoclonal antibodies (MAbs).

[0082]The term "agent" is used herein to denote a chemical compound, a
mixture of chemical compounds, a biological molecule, a biological
macromolecule, or an extract made from biological materials.

[0083]The term "stabilizing agent" refers to an agent that stabilizes a
specific binding agent in a composition. A specific binding agent is
"stabilized" in a composition if the specific binding agent retains more
of its physical stability and/or chemical stability and/or biological
activity in a composition comprising a stabilizing agent compared with
the composition not comprising the stabilizing agent.

[0084]A specific binding agent in a composition contained in a container,
e.g., a syringe, is "stabilized" if the specific binding agent retains at
least the same or similar physical stability and/or chemical stability
and/or biological activity after being subjected to one or more of the
laboratory tests which simulate shipping conditions that are discussed in
the following documents: Singh, J., S. P. Singh and G. Burgess,
Measurement and Analysis of US Truck Vibration for Leaf Spring and Air
Ride Suspensions and Development of Test Tests, Packaging Technology and
Science, Vol 19, 2006; International Safe Transit Association (ISTA)
Resource Book 2006, Test Procedure 3A; Singh, S. P. and J. Marcondes,
"Vibration Levels in Commercial Truck Shipments as a Function of
Suspension and Payload", Journal of Testing and Evaluation, ASTM, Vol 20,
No. 6, 466-469, 1992; Kipp, William I., Vibration Testing Equivalence,
How Many Hours Of Testing Equals How Many Miles Of Transport?,
Proceedings of ISTA Con 2000; Rouillard, V., A New Approach to Analyzing
and Simulating Shock and Vibration, Proceedings of Dimensions 06,
International Safe Transit Association, East Lansing, Mich. 48823, 2006;
Singh, S. P., G. Burgess and P. Rojuckarin, "Test Protocol for Simulating
Truck and Rail Vibration and Rail Impacts in Shipments of Automotive
Engine Racks", Packaging Technology and Science, Vol. 8, 33-41, 1995;
Brandenburg and Lee. (2001). Fundamentals of Packaging Dynamics. L.A.B.
Equipment Inc.: Skaneateles, N.Y.; and Singh, S. P., G. Burgess,
Marcondes, Jorge A., and Antle, John R., "Measuring the Package Shipping
Environment in Refrigerated Ocean Vessels", Packaging Technology and
Science, Vol. 6, 175-181, 1993. A specific binding agent in a composition
contained in a container is considered "stabilized" if the specific
binding agent retains at least the same or similar physical stability
and/or chemical stability and/or biological stability after being
subjected to at least one test, even if one or more of those properties
is not retained after being subjected to one or more tests. In certain
embodiments, the laboratory test includes vibration, shock/drop, and/or
pressure changes to simulate air and/or vehicular travel. The laboratory
test also includes a control, in which the specific binding agent
contained in a container is not subjected to vibration, shock/drop,
and/or pressure changes. Following vibration, shock/drop, and/or pressure
changes to simulate air and/or vehicular travel, the physical stability
and/or chemical stability and/or biological activity of the specific
binding agent is determined and compared to the physical stability and/or
chemical stability and/or biological activity of the control specific
binding agent. In certain embodiments, a specific binding agent is
considered to retain at least the same or similar physical stability
and/or chemical stability and/or biological activity if the specific
binding agent is suitable for use as a pharmaceutical in a human.

[0085]When a stabilizing agent is used, the phrase "retains its physical
stability" means that a specific binding agent in a composition shows
less aggregation and/or precipitation and/or denaturation in a
composition comprising a stabilizing agent compared with the composition
not comprising the stabilizing agent. The phrase "retains its physical
stability" also means a specific binding agent in a composition contained
in a container of a certain type, e.g., syringe, shows the same or
similar or less aggregation and/or precipitation and/or denaturation
after being subjected to one or more of the laboratory tests discussed in
paragraph 84, which simulate shipping conditions. A specific binding
agent in a composition contained in a container is considered to retain
its physical stability if the specific binding agent shows the same or
similar or less aggregation and/or precipitation and/or denaturation
after being subjected to at least one test, even if one or more of those
properties is not the same or similar or less after being subjected to
one or more tests. In certain embodiments, a composition contained in a
container of a certain type, e.g., syringe, shows the same or similar or
less aggregation and/or precipitation and/or denaturation after being
subjected to a laboratory test which simulates shipping conditions,
followed by subsequent storage under static conditions as the specific
binding agent in the composition contained in a container of the same
type stored under static conditions and not subjected to a laboratory
test which simulates shipping conditions. In certain embodiments, a
composition contained in a container subjected to a laboratory test which
simulates shipping conditions, and followed by subsequent storage under
static conditions is stored at a temperature between 2° C. and
8° C. In certain embodiments, a composition contained in a
container subjected to a laboratory test which simulates shipping
conditions, and followed by subsequent storage under static conditions is
stored at a temperature between 15° C. and 45° C. In
certain embodiments, a composition contained in a container stored under
static conditions and not subjected to a laboratory test which simulates
shipping conditions, is stored in a freezer at a temperature between
-20° C. and -80° C. In certain embodiments, a composition
contained in a container subjected to a laboratory test which simulates
shipping conditions, and followed by subsequent storage under static
conditions is stored for at least 1 month to at least 24 months.
Exemplary storage periods include, but are not limited to, at least 1
month, at least 3 months, at least 6 months, at least 9 months, at least
12 months, at least 18 months, and at least 24 months. Exemplary methods
of determining the amount of aggregation and/or precipitation and/or
denaturation of a specific binding agent include, but are not limited to,
visual inspection; subvisible particulate counting by light obscuration,
for example, by using a HIAC (Royco) instrument; microscopic particle
counting; size-exclusion high-performance liquid chromatography
(SEC-HPLC), and SDS-PAGE. A specific binding agent in a composition
contained in a container is considered to retain its physical stability
if the specific binding agent shows the same or similar or less
aggregation and/or precipitation and/or denaturation as determined in at
least one of those determining methods, even if one or more of those
properties is not the same or similar or less as determined in one or
more of those determining methods. In certain embodiments, a specific
binding agent is considered to show the same or similar or less
aggregation and/or precipitation and/or denaturation if the specific
binding agent is suitable for use as a pharmaceutical in a human.

[0086]When a stabilizing agent is used, the phrase "retains its chemical
stability" means that a specific binding agent in a composition shows
less chemical alteration in a composition comprising a stabilizing agent
compared with the composition not comprising the stabilizing agent. The
phrase "retains its chemical stability" also means a specific binding
agent in a composition contained in a container of a certain type, e.g.,
syringe, shows the same or similar or less chemical alteration after
being subjected to one or more of the laboratory tests discussed in
paragraph 84, which simulate shipping conditions. Examples of chemical
alteration include, but are not limited to, size modification, for
example, including, but not limited to, clipping. Clipping refers to
cleavage of a specific binding agent that results in smaller fragments.
In certain embodiments, clipping is a result of proteolysis. Examples of
chemical alteration include, but are not limited to, charge alteration,
for example, including, but not limited to, deamidation. Examples of
chemical alteration include, but are not limited to,
hydrophilic/hydrophobic alteration, for example, including, but not
limited to, oxidation. Examples of chemical alteration include, but are
not limited to, isomerization. A specific binding agent in a composition
contained in a container is considered to retain its chemical stability
if the specific binding agent shows the same or similar or less of any
type of chemical alteration after being subjected to at least one test,
even if one or more types of chemical alteration is not the same or
similar or less after being subjected to one or more tests. In certain
embodiments, a composition contained in a container of a certain type,
e.g., syringe, shows the same or similar or less chemical alteration
after being subjected to a laboratory test which simulates shipping
conditions, followed by subsequent storage under static conditions as the
specific binding agent in the composition contained in a container of the
same type stored under static conditions and not subjected to a
laboratory test which simulates shipping conditions. In certain
embodiments, a composition contained in a container subjected to a
laboratory test which simulates shipping conditions, and followed by
subsequent storage under static conditions is stored at a temperature
between 2° C. and 8° C. In certain embodiments, a
composition contained in a container subjected to a laboratory test which
simulates shipping conditions, and followed by subsequent storage under
static conditions is stored at a temperature between 15° C. and
45° C. In certain embodiments, a composition contained in a
container stored under static conditions and not subjected to a
laboratory test which simulates shipping conditions, is stored in a
freezer at a temperature between -20° C. and -80° C. In
certain embodiments, a composition contained in a container subjected to
a laboratory test which simulates shipping conditions, and followed by
subsequent storage under static conditions is stored for at least 1 month
to at least 24 months. Exemplary storage periods include, but are not
limited to, at least 1 month, at least 3 months, at least 6 months, at
least 9 months, at least 12 months, at least 18 months, and at least 24
months. Exemplary methods of determining the amount of chemical
alteration of a specific binding agent include, but are not limited to,
cation-exchange HPLC, reversed-phase HPLC, SDS-PAGE, and peptide mapping.
A specific binding agent in a composition contained in a container is
considered to retain its chemical stability if the specific binding agent
shows the same or similar or less of any type of chemical alteration as
determined in at least one of those determining methods, even if one or
more types of chemical alteration is not the same or similar or less as
determined in one or more of those determining methods. In certain
embodiments, a specific binding agent is considered to show the same or
similar or less chemical alteration if the specific binding agent is
suitable for use as a pharmaceutical in a human.

[0087]When a stabilizing agent is used, the phrase "retains its biological
activity" means that a specific binding agent in a composition
demonstrates more biological activity at a given time after the
composition was prepared in a composition comprising a stabilizing agent
compared with the composition not comprising the stabilizing agent. The
phrase "retains its biological activity" also means a specific binding
agent in a composition contained in a container of a certain type, e.g.,
syringe, demonstrates at least the same or similar biological activity at
a given time after the composition was prepared and after being subjected
to one or more of the laboratory tests discussed in paragraph 84, which
simulate shipping conditions. A specific binding agent in a composition
contained in a container is considered to retain its biological activity
if the specific binding agent demonstrates at least the same or similar
of any type of biological activity at a given time after the composition
was prepared and after being subjected to at least one test, even if one
or more types of biological activity is not at least the same or similar
at a given time after the composition was prepared and after being
subjected to one or more tests. In certain embodiments, a composition
contained in a container of a certain type, e.g., syringe, demonstrates
at least the same or similar biological activity at a given time after
the composition was prepared and after being subjected to a laboratory
test which simulates shipping conditions, followed by subsequent storage
under static conditions as the specific binding agent in the composition
contained in a container of the same type stored under static conditions
and not subjected to a laboratory test which simulates shipping
conditions. In certain embodiments, a composition contained in a
container subjected to a laboratory test which simulates shipping
conditions, and followed by subsequent storage under static conditions is
stored at a temperature between 2° C. and 8° C. In certain
embodiments, a composition contained in a container subjected to a
laboratory test which simulates shipping conditions, and followed by
subsequent storage under static conditions is stored at a temperature
between 15° C. and 45° C. In certain embodiments, a
composition contained in a container stored under static conditions and
not subjected to a laboratory test which simulates shipping conditions,
is stored in a freezer at a temperature between -20° C. and
-80° C. In certain embodiments, a composition contained in a
container subjected to a laboratory test which simulates shipping
conditions, and followed by subsequent storage under static conditions is
stored for at least 1 month to at least 24 months. Exemplary storage
periods include, but are not limited to, at least 1 month, at least 3
months, at least 6 months, at least 9 months, at least 12 months, at
least 18 months, and at least 24 months. In certain embodiments,
biological activity is determined by an assay appropriate for determining
biological activity. Exemplary assays to determine biological activity of
a specific binding agent include, but are not limited to, antigen binding
assays and receptor phosphorylation assays. Exemplary antigen binding
assays include, but are not limited to, ELISA assays, immunoprecipitation
assays, and affinity assays, for example, including, but not limited to,
BIAcore® assays. Certain exemplary methods and assays to determine
biological activity of specific binding agents to RANKL have been
described, e.g., in U.S. Publication No. 2004/0033535, published Feb. 19,
2004. Certain exemplary methods and assays to determine biological
activity of specific binding agents to IL-1R1 have been described, e.g.,
in U.S. Publication No. 2004/0097712, published May 20, 2004. Certain
exemplary methods and assays to determine biological activity of specific
binding agents to TNF and of specific binding agents to TNF-R have been
described, e.g., in U.S. Pat. No. 5,945,397. A specific binding agent in
a composition contained in a container is considered to retain its
biological activity if the specific binding agent demonstrates at least
the same or similar of any type of biological activity at a given time
after the composition was prepared as determined in at least one assay,
even if one or more types of biological activity at a given time after
the composition was prepared is not the same or similar as determined in
one or more assays. In certain embodiments, a specific binding agent is
considered to demonstrate at least the same or similar biological
activity at a given time after the composition was prepared if the
specific binding agent is suitable for use as a pharmaceutical in a
human.

[0089]"Aggregation" refers to the formation of multimers of individual
protein molecules through non-covalent or covalent interactions.
Aggregation also refers to the formation of particles. Particles may be
either subvisible or visible. Subvisible particles are of a size between
2 μM and 100 μM. Visible particles are of a size greater than 100
μM. Aggregation can be reversible or irreversible. In certain
instances, when the loss of tertiary structure or partial unfolding
occurs, hydrophobic amino acid residues which are typically hidden within
the folded protein structure are exposed to the solution. In certain
instances, this promotes hydrophobic-hydrophobic interactions between
individual protein molecules, resulting in aggregation. Srisailam et al.,
J Am Chem Soc 124 (9):1884-8 (2002), for example, has determined that
certain conformational changes of a protein accompany aggregation, and
that certain regions of specific proteins can be identified as
particularly responsible for the formation of aggregates. In certain
instances, protein aggregation can be induced by heat (Sun et al., J
Agric Food Chem 50(6): 1636-42 (2002)), organic solvents (Srisailam et
al., supra), and reagents such as SDS and lysophospholipids (Hagihara et
al., Biochem 41(3): 1020-6 (2002)). Aggregation can be a significant
problem in in vitro protein purification and formulation. In certain
instances, after formation of aggregates, solubilization with strong
denaturating solutions followed by renaturation and proper refolding may
be needed before biological activity is restored.

[0090]"Denaturation" refers to an alteration of the three-dimensional
structure of a polypeptide. Three-dimensional structure of a polypeptide
includes, but is not limited to, secondary structure and tertiary
structure. Secondary structure refers to the local conformation of a
portion of a polypeptide. Certain exemplary secondary structures include,
but are not limited to, α helix; β conformation, β sheet,
and β turn. Tertiary structure refers to the overall
three-dimensional arrangement of atoms in a polypeptide. In certain
instances, tertiary structure includes interactions between amino acids
that are located far apart in the linear sequence. In certain instances,
the alteration of three-dimensional structure is such that a polypeptide
is partially or completely unfolded. In certain instances, the alteration
of three-dimensional structure is sufficient to cause a partial or
complete loss of function. In certain instances, denaturation can be
induced by exposure of a polypeptide to any one or more of the following:
heat; pH extremes; organic solvents, including, but not limited to,
alcohol and acetone; detergents, including, but not limited to, SDS; and
chaotropic reagents, including, but not limited to, urea and guanidine
hydrochloride. In certain instances, denaturation of a polypeptide can be
induced by exposure of the polypeptide to a surface of a container, for
example, including, but not limited to, containers comprising glass,
stainless steel, polycarbonate, polytetrafluoroethylene (Teflon®),
polyurethane, silicone, polyvinyl chloride, ethylene-vinyl acetate,
polyester, and polyolefin. In certain instances, denaturation of a
polypeptide can be induced by exposure of the polypeptide to a surface of
a container closure, for example, including, but not limited to,
container closures comprising silicone oil, butyl rubber, fluorocarbon
and tungsten. In certain instances, denaturation of a polypeptide can be
induced by exposure of the polypeptide to a phase interface, for example,
including, but not limited to an air/liquid interface, an ice/liquid
interface, and an aqueous/oil interface. In certain instances,
denaturation of certain polypeptides, for example, globular proteins, by
exposure to organic solvents, urea, and detergents results in disruption
of hydrophobic interactions within the polypeptide. In certain instances,
denaturation of a polypeptide by exposure to, for example, pH extremes
results in alteration of the net charge of the polypeptide, which causes
electrostatic repulsion and disruption of certain hydrogen bonding within
the polypeptide.

[0091]The term "shipping," "ships," or "shipped" refers to transporting a
composition in a container in a vehicle, airplane, and/or ship, by any
route, for any distance, and at any temperature.

[0092]The phrase "stored under static conditions," or "static storage
conditions" refers to keeping a composition in a container in a location
without shipping.

[0093]The term "headspace" refers to the space between a liquid in a
container and the container closure. See, e.g., FIG. 5 (B) and FIGS. 6
(A) and (B). In certain embodiments, the headspace is of a size such that
a meniscus is formed by the liquid in the container, which is visible by
eye or by light microscopy. As used herein, a "meniscus" refers to a
concave upper surface of a liquid in a container. When the container is
in a vertical (upright) position, the meniscus extends across the
diameter of a container and no liquid touches the bottom surface of the
container closure. In certain embodiments, the headspace is the distance
between the top of the meniscus and the bottom surface of the container
closure, e.g., the flat body portion in the center of a plunger. In
certain embodiments, the headspace is of a size such that a meniscus is
not formed, but is of a size such that an air bubble is formed by the
liquid in the container, which is visible by eye or by light microscopy.
As used herein, an "air bubble" does not extend across the diameter of a
container when the container is in a vertical position, and some, but not
all, of the liquid touches the bottom surface of the container closure.
In certain embodiments, the air bubble is spherical in shape. In certain
of those embodiments, the headspace is the diameter of the air bubble. In
certain embodiments, the air bubble is not spherical in shape. In certain
such embodiments, the air bubble is elliptical in shape. In certain of
those embodiments, the headspace is the largest dimension of the air
bubble. In certain embodiments, headspace is measured using calipers. In
certain such embodiments, a 10× magnifying lens is used with a
certified and calibrated caliper. An exemplary caliper is Mitutoyo Series
500, MCN number 900-G1-222. In certain embodiments, headspace is measured
using a microscope and microscope ruler. In certain such embodiments,
calipers are used to record the distance between the top of the meniscus
to the bottom of the flat body of the plunger using calipers. In certain
embodiments, the headspace of a prefilled and stoppered syringe is
measured with an optical comparator. An exemplary optical comparator is
Deltronic DH 216, Horizontal Optical Comparator. In certain such
embodiments, measurements are made by placing the syringe in a vertical
position and parallel to the optical lens. A magnified image is projected
onto a screen for inspection. Calipers on the optical comparator are used
to record the distance between the top of the meniscus to the bottom of
the flat body of the plunger. In certain embodiments, the headspace is
the distance in millimeters from the top of the meniscus to the bottom of
the flat body of the plunger.

[0094]As used herein, headspace is "minimized" when the headspace
measurement is 3.0 mm or less using the caliper and/or microscope
measurement methods described above in paragraph 90 and in Example 2
below. A headspace is considered "minimized" if the headspace measurement
is 3.0 mm or less using at least one measurement method, even if the
measurement is greater than 3.0 mm using one or more other tests. Certain
exemplary minimized headspace measurements include, but are not limited
to, 2.7 mm or less, or 2.5 mm or less, or 2 mm or less, or 1.5 mm or
less, or 1 mm or less, or 0.5 mm or less, or 0.2 mm or less, or 0.1 mm or
less, or no detectable headspace. In certain embodiments, the minimized
headspace measurement is between 2.5 mm and 3.0 mm, or 2.0 mm and 2.5 mm,
or 1.5 mm and 2.0 mm, or 1.0 and 1.5 mm. See, e.g., FIG. 6 (B). In
certain embodiments, headspace is minimized when the headspace cannot be
measured using the caliper and/or microscope measurement methods
described herein. In certain such embodiments, there is no meniscus
visible by eye or by light microscopy. In certain such embodiments, there
is no air bubble visible by eye or by light microscopy.

[0095]A "container closure" refers to a part of a container or container
assembly that covers or seals the container. In certain embodiments, the
container closure holds a composition inside a container. In certain
embodiments, the container closure is impermeable to microbial ingress.
Exemplary container closures include, but are not limited to, caps, lids,
plungers, and stoppers.

[0096]A "prefilled syringe" refers to a container for a composition, for
example, a therapeutic composition, in which the container is a syringe,
the composition is placed in the syringe prior to administration of the
composition to a patient, and the syringe is covered with a syringe
closure, for example, but not limited to, a plunger. In certain
embodiments, the composition is placed in the syringe in a manufacturing
fill facility. In certain embodiments, the syringe is washed and
sterilized prior to placing the composition in the syringe. In certain
embodiments, the prefilled syringe includes the composition for at least
1 day, or at least 7 days, or at least 14 days, or at least 1 month, or
at least 6 months, or at least 1 year, or at least 2 years prior to
administration of the composition to a patient. In certain embodiments,
the prefilled syringe is subject to storage and/or shipping conditions.

[0097]The term "silicone" refers to a lubricant comprising a
semi-inorganic polymer based on the structural unit R2SiO, where R
is an organic group. In certain embodiments, the silicone is
polydimethylsiloxane, also referred to as silicone oil. In certain
embodiments, the internal surface of a syringe barrel, the surface of a
syringe plunger, and/or the surface of a syringe needle is coated with
silicone. In certain embodiments, other types of containers and/or
container closures, including, but not limited to, stopcocks, are coated
with silicone. Certain exemplary polydimethylsiloxanes include, but are
not limited to, Dow Corning® 360 Medical Fluid, including for
example, but not limited to, Dow Corning® 360 Medical Fluid having a
viscosity of 350 centistokes, Dow Corning® 360 Medical Fluid having a
viscosity of 1000 centistokes, Dow Corning® 360 Medical Fluid having
a viscosity of 12,500 centistokes, and Dow Corning® MDX4-4159 fluid.
In certain embodiments, silicone oil is sprayed on the surface. In
certain embodiments, silicone oil is wiped on the surface. In certain
embodiments, silicone oil is baked. In certain embodiments, silicone oil
is cross-linked.

[0099]The term "baked silicone" refers to silicone, which, after being
applied to a container, for example, including, but not limited to, a
syringe, is treated with heat thereby promoting binding of silicone to
the surface of the container.

[0100]The term "cross-linked silicone" refers to a cross-linkable silicone
oil which has been subjected to a cross-linking treatment. Cross-linkable
silicone oils include, but are not limited to, silicone oils having
reactive and/or functional chemical groups enabling cross-linking of the
oil. An exemplary commercially available cross-linkable silicone oil
includes, but is not limited to, Dow Corning® MDX4-4159. Exemplary
cross-linking treatments include, but are not limited to, treatment by
irradiation, including for example, but not limited to, exposure to an
electron, x-ray, or γ-ray source; and treatment in an ionizing
plasma, including for example, but not limited to, oxygen plasma.

[0101]The terms "silicone-free material" and "material lacks silicone"
refers to material used in the manufacture of a container or a container
closure in which no silicone has been added to coat a surface. In certain
embodiments, silicone is not detectable as determined in one or more of
the following tests: exposing the material to solvent that will extract
silicone, and detecting silicone by either (1) an Inductively Coupled
Plasma (ICP) assay coupled with Mass Spectrometry (ICP-MS), atomic
emission spectroscopy (ICP-AES), or atomic absorption (ICP-AA), as
described in Kennan J J, Breen L L, Lane T H, Taylor R B., Methods for
detecting silicones in biological matrixes, Analytical Chemistry,
71(15):3054-60, 1999; Mundry T, Surmann P, Schurreit T., Trace analysis
of silicone oil in aqueous parenteral formulation and glass containers by
graphite furnace atomic absorption spectrometry, Drugs made in Germany,
Vol 44, No 2, 47-56, 2001; Carter, J., L. Ebdon, and E. H. Evans,
Speciation of silicon and phosphorous using liquid chromatography coupled
with sector field high resolution ICP-MS, Microchemical Journal, 2004,
76(1-2): p. 35-41; or Klemens P, Heumann K G., Development of an
ICP-HRIDMS method for accurate determination of traces of silicon in
biological and clinical samples, Fresenius J Anal Chem, 371:758-763,
2001; or (2) a Fourier Transform Infrared (FTIR) spectroscopic assay, as
described in Silverstein, R. M., Bassler, G. C., Morrill, T. C.
Spectrometric Identification of Organic Compounds, 5th Ed., 1991; or
Gungel, H., Menceoglu, Yildiz, B., Akbulut, O., Fourier Transform
Infrared And 1H Nuclear Magnetic Resonance Spectroscopic Findings Of
Silicone Oil Removed From Eyes And The Relationship Of Emulsification
With Retinotomy And Glaucoma, The Journal Of Retinal And Vitreous
Diseases, Vol 25, No 3, 332-338, 2005. Silicon is considered not to be
detectable if it is not detectable in at least one of these tests, even
if it is detectable in one or more other tests.

[0102]The terms "lubricant-free material" and "material lacks lubricant"
refers to material used in the manufacture of a container or a container
closure in which no lubricant has been added to coat a surface. In
certain embodiments, lubricant is not detectable as determined in one or
more of the following tests: exposing the material to solvent that will
extract lubricant, and detecting lubricant by either (1) an Inductively
Coupled Plasma (ICP) assay coupled with Mass Spectrometry (ICP-MS),
atomic emission spectroscopy (ICP-AES), or atomic absorption (ICP-AA), as
described in Kennan J J, Breen L L, Lane T H, Taylor R B., Methods for
detecting silicones in biological matrixes, Analytical Chemistry,
71(15):3054-60, 1999; Mundry T, Surmann P, Schurreit T., Trace analysis
of silicone oil in aqueous parenteral formulation and glass containers by
graphite furnace atomic absorption spectrometry, Drugs made in Germany,
Vol 44, No 2, 47-56, 2001; Carter, J., L. Ebdon, and E. H. Evans,
Speciation of silicon and phosphorous using liquid chromatography coupled
with sector field high resolution ICP-MS, Microchemical Journal, 2004,
76(1-2): p. 35-41; or Klemens P, Heumann K G., Development of an
ICP-HRIDMS method for accurate determination of traces of silicon in
biological and clinical samples, Fresenius J Anal Chem, 371:758-763,
2001; or (2) a Fourier Transform Infrared (FTIR) spectroscopic assay, as
described in Silverstein, R. M., Bassler, G. C., Morrill, T. C.
Spectrometric Identification of Organic Compounds, 5th Ed., 1991; or
Gungel, H., Menceoglu, Yildiz, B., Akbulut, O., Fourier Transform
Infrared And 1H Nuclear Magnetic Resonance Spectroscopic Findings Of
Silicone Oil Removed From Eyes And The Relationship Of Emulsification
With Retinotomy And Glaucoma, The Journal Of Retinal And Vitreous
Diseases, Vol 25, No 3, 332-338, 2005. Lubricant is considered not to be
detectable if it is not detectable in at least one of these tests, even
if it is detectable in one or more other tests.

[0104]A "buffering agent" or "buffer" refers to an agent that maintains
the pH of a composition within a desired range.

[0105]The terms "osteopenic disorder," "bone loss," or "bone loss
condition" includes, but is not limited to, osteoporosis; including, but
not limited to, postmenopausal osteoporosis, endocrine osteoporosis
(including, but not limited to, hyperthyroidism, hyperparathyroidism,
Cushing's syndrome, and acromegaly), hereditary and congenital forms of
osteoporosis (including, but not limited to, osteogenesis imperfecta,
homocystinuria, Menkes' syndrome, and Riley-Day syndrome); and
osteoporosis due to immobilization of extremities; Paget's disease of
bone (osteitis deformans) in adults and juveniles; osteomyelitis, or an
infectious lesion in bone, leading to bone loss; hypercalcemia resulting
from solid tumors (including, but not limited to, breast, lung and
kidney) and hematologic malignancies (including, but not limited to,
multiple myeloma, lymphoma and leukemia), idiopathic hypercalcemia, and
hypercalcemia associated with hyperthyroidism and renal function
disorders; osteopenia following surgery, associated with use of steroids,
such as glucocorticoids, and associated with disorders of the small and
large intestine and with chronic hepatic and renal diseases;
osteonecrosis, or bone cell death, associated with traumatic injury or
nontraumatic necrosis; bone loss associated with anemia or inflammatory
or autoimmune conditions such as systemic lupus erythematosus and
rheumatoid arthritis, and periodontal disease.

[0106]In addition to those bone loss conditions, certain cancers,
including those which metastasize to bone or are resident in bone are
known to increase osteoclast activity and induce bone resorption. Such
cancers include, but are not limited to, breast cancer, prostate cancer,
and multiple myeloma. In certain instances, these cancers are known to
produce factors that result in the over-expression of RANKL in the bone,
and lead to increased osteoclast numbers and activity. Accordingly, bone
loss disorders include, but are not limited to, breast cancer, prostate
cancer, and solid tumors that have metastasized to bone or are capable of
metastasizing to bone; multiple myeloma; and giant cell tumor of bone.
Other bone loss conditions include, but are not limited to,
chemotherapy-induced bone loss in patients with metastatic and
non-metastatic cancer, including, but not limited to, breast cancer and
prostate cancer. In certain instances, bone loss occurs during hormone
ablative therapy, such as, for example, but not limited, with adjuvant
aromatase inhibitors.

[0107]A disease or medical condition is considered to be an "interleukin-1
(IL-1) mediated disease" if the naturally-occurring or
experimentally-induced disease or medical condition is associated with
elevated levels of IL-1 in bodily fluids or tissue or if cells or tissues
taken from the body produce elevated levels of IL-1 in culture. Elevated
levels of IL-1 include, for example, but are not limited to, levels that
exceed those normally found in a particular cell or tissue; and any
detectable level of IL-1 in a cell or tissue that normally does not
express a detectable level of IL-1. In certain instances, IL-1 mediated
diseases are also recognized by either one or both of the following
additional two conditions: (1) pathological findings associated with the
disease or medical condition mimicked experimentally in animals by
administration of IL-1 or by experimental conditions resulting in
up-regulation of expression of IL-1; and (2) a pathology induced in
experimental animal models of the disease or medical condition inhibited
or abolished by treatment with agents that inhibit the action of IL-1. In
certain IL-1 mediated diseases, at least two of the three conditions are
met. In certain IL-1 mediated diseases, all three conditions are met.

[0109]A disease or medical condition is considered to be an "TNF-mediated
disease" if the naturally-occurring or experimentally-induced disease or
medical condition is associated with elevated levels of TNF in bodily
fluids or tissue or if cells or tissues taken from the body produce
elevated levels of TNF in culture. Elevated levels of TNF include, for
example, but are not limited to, levels that exceed those normally found
in a particular cell or tissue; and any detectable level of TNF in a cell
or tissue that normally does not express a detectable level of TNF. In
certain instances, TNF-mediated diseases are also recognized by either
one or both of the following additional two conditions: (1) pathological
findings associated with the disease or medical condition mimicked
experimentally in animals by administration of TNF or by experimental
conditions resulting in up-regulation of expression of TNF; and (2) a
pathology induced in experimental animal models of the disease or medical
condition inhibited or abolished by treatment with agents that inhibit
the action of TNF. In certain TNF-mediated diseases, at least two of the
three conditions are met. In certain TNF-mediated diseases, all three
conditions are met.

[0111]A specific binding agent "substantially inhibits binding" of a
ligand to a receptor when an excess of specific binding agent reduces the
quantity of receptor bound to the ligand by at least about 20%, 40%, 60%,
80%, 85%, or more (as measured in an in vitro competitive binding assay).
In certain embodiments, a specific binding agent is an antibody. In
certain such embodiments, an antibody substantially inhibits binding of
RANKL to RANK, or substantially inhibits binding of IL-1 to IL-1R1. In
certain embodiments, a specific binding agent is a soluble fusion
polypeptide. In certain such embodiments, a soluble fusion polypeptide
substantially inhibits binding of TNF to TNF-R.

[0113]The term "pharmaceutical agent or drug" as used herein refers to a
chemical compound or composition capable of inducing a desired
therapeutic effect when properly administered to a patient. As used
herein, a therapeutic effect may or may not include a prophylactic
effect.

[0114]The term "modulator," as used herein, is a compound that changes or
alters the activity or function of a molecule. For example, a modulator
may cause an increase or decrease in the magnitude of a certain activity
or function of a molecule compared to the magnitude of the activity or
function observed in the absence of the modulator. In certain
embodiments, a modulator is an inhibitor, which decreases the magnitude
of at least one activity or function of a molecule. Certain exemplary
activities and functions of a molecule include, but are not limited to,
binding affinity, enzymatic activity, and signal transduction. Certain
exemplary inhibitors include, but are not limited to, proteins, peptides,
antibodies, peptibodies, carbohydrates or small organic molecules.
Peptibodies are described in, e.g., U.S. Pat. No. 6,660,843 and PCT
Publication No. WO 01/83525.

[0115]As used herein, "substantially pure" means an object species is the
predominant species present (i.e., on a molar basis it is more abundant
than any other individual species in the composition). In certain
embodiments, a substantially purified fraction is a composition wherein
the object species comprises at least about 50 percent (on a molar basis)
of all macromolecular species present. In certain embodiments, a
substantially pure composition will comprise more than about 80%, 85%,
90%, 95%, or 99% of all macromolecular species present in the
composition. In certain embodiments, the object species is purified to
essential homogeneity (contaminant species cannot be detected in the
composition by conventional detection methods) wherein the composition
consists essentially of a single macromolecular species.

[0116]The term "patient" includes human and animal subjects.

Certain Exemplary Specific Binding Agents

[0117]In certain instances, TNF is released by activated macrophages and T
cells, inducing a wide variety of effects on a large number of cell
types. In certain instances, TNF plays a role in regulating the normal
immune response, as well as in various pathological and disease states.
Certain such pathological and disease states include, but are not limited
to, systemic toxicity associated with sepsis, pathogenesis of AIDS, and
various autoimmune and inflammatory diseases, including, but not limited
to, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing
spondylitis, and plaque psoriasis.

[0118]TNF proteins initiate their biological effect on cells, in certain
instances, by binding to specific TNF receptor (TNF-R) proteins expressed
on the plasma membrane of a TNF-responsive cell. Therefore, in certain
instances, a reduction in TNF-mediated cellular responses may reduce the
severity of arthritic, immune, autoimmune, and/or inflammatory disorders.
According to certain embodiments, specific binding agents to TNF are used
to treat immune, autoimmune, and/or inflammatory disorders, including,
but not limited to, those mentioned above.

[0121]Certain exemplary soluble TNF-R and soluble TNF-R fusion
polypeptides and methods of making and using such polypeptides are
described in U.S. Pat. No. 5,945,397 and Mohler et al., J. Immunol.
151:1548-1561 (1993). In certain such embodiments, a purified soluble
human TNF-R fusion polypeptide is provided. In certain such embodiments,
a purified soluble human TNF-R fusion polypeptide is sTNFR:Fc as
described in Mohler et al., J. Immunol. 151:1548-1561 (1993), or
etanercept, which is sold under the tradename Enbrel®, discussed in
the Examples below.

[0122]In certain instances, RANKL is involved in the formation of
osteoclasts. In certain instances, RANKL binds to a receptor, RANK, which
increases osteoclast activity. In certain instances, increased osteoclast
activity correlates with certain osteopenic disorders, including
post-menopausal osteoporosis, Paget's disease, lytic bone metastases, and
rheumatoid arthritis. Therefore, in certain instances, a reduction in
RANKL activity may result in a decrease in osteoclast activity and may
reduce the severity of osteopenic disorders. According to certain
embodiments, specific binding agents to RANKL are used treat osteopenic
disorders, including by not limited to, those mentioned above.

[0123]In certain embodiments, specific binding agents to RANKL are fully
human monoclonal antibodies. In certain embodiments, nucleotide sequences
encoding heavy and light chain immunoglobulin molecules, and
corresponding amino acid sequences, particularly sequences corresponding
to the variable regions are provided. In certain embodiments, sequences
corresponding to complementarity determining regions (CDR's),
specifically from CDR1 through CDR3, are provided. According to certain
embodiments, a hybridoma cell line expressing such an immunoglobulin
molecule is provided. According to certain embodiments, a hybridoma cell
line expressing such a monoclonal antibody is provided. According to
certain embodiments, a Chinese Hamster Ovary (CHO) cell line expressing
such a monoclonal antibody is provided. In certain embodiments, a
purified human monoclonal antibody to human RANKL is provided.

[0124]Certain exemplary antibodies to RANKL (also referred to as OPGL) and
methods of making and using such antibodies are described in U.S.
Publication No. 2004/0033535, published Feb. 19, 2004. In certain such
embodiments, a purified human monoclonal antibody to human RANKL is
provided. See, e.g., U.S. Publication No. 2004/0033535. In certain such
embodiments, a purified human monoclonal antibody to human RANKL is
αRANKL-1, discussed in the Examples below.

[0125]In certain instances, IL-1, a cytokine, is involved in the
inflammatory response. In certain instances, IL-1 binds to a receptor,
IL-1R1, followed by binding to IL-1RAcP. Those events are followed by
signal transduction resulting in the induction of a cellular response,
which, in certain instances, leads to inflammation. In certain instances,
inflammation is associated with injuries resulting from mechanical
damage, infection, or antigenic stimulation. In certain instances,
inflammatory reactions are expressed pathologically. Such conditions
arise, in certain instances, when the inflammation is expressed in an
exaggerated manner, is inappropriately stimulated, or persists after the
injurious agent is removed. Exemplary pathological conditions mediated by
IL-1 include, but are not limited to, rheumatoid arthritis and
osteoarthritis. Therefore, in certain instances, a reduction in IL-1
mediated signal transduction activity may result in a decrease in
cellular responses leading to inflammation and may reduce the severity of
arthritic and other inflammatory disorders. According to certain
embodiments, specific binding agents to IL-1R1 are used treat
inflammatory disorders, including by not limited to, those mentioned
above.

[0126]In certain embodiments, specific binding agents to IL-1R1 are fully
human monoclonal antibodies. In certain embodiments, nucleotide sequences
encoding heavy and light chain immunoglobulin molecules, and
corresponding amino acid sequences, particularly sequences corresponding
to the variable regions are provided. In certain embodiments, sequences
corresponding to complementarity determining regions (CDR's),
specifically from CDR1 through CDR3, are provided. According to certain
embodiments, a hybridoma cell line expressing such an immunoglobulin
molecule is provided. According to certain embodiments, a hybridoma cell
line expressing such a monoclonal antibody is provided. According to
certain embodiments, a Chinese Hamster Ovary (CHO) cell line expressing
such a monoclonal antibody is provided. In certain embodiments, a
monoclonal antibody is selected from at least one of 15C4, 26F5, and
27F2. In certain embodiments, a purified human monoclonal antibody to
human IL-1R1 is provided.

[0127]Certain exemplary antibodies to IL-1R1 and methods of making and
using such antibodies are described in U.S. Publication No. 2004/0097712,
published May 20, 2004. In certain such embodiments, a purified human
monoclonal antibody to human IL-1R1 is provided. In certain such
embodiments, a purified human monoclonal antibody to human IL-1R1 has a
light chain variable region of SEQ ID NO:12 and a heavy chain variable
region of SEQ ID NO:10 as set forth in U.S. Publication No. 2004/0097712;
or alternatively a light chain variable region of SEQ ID NO:12 and a
heavy chain variable region of SEQ ID NO:14 as set forth in U.S.
Publication No. 2004/0097712; or alternatively a light chain variable
region of SEQ ID NO:18 and a heavy chain variable region of SEQ ID NO:16
as set forth in U.S. Publication No. 2004/0097712.

[0128]In certain embodiments, a human monoclonal antibody to human RANKL
and/or a human monoclonal antibody to IL-1R1 is a fully human monoclonal
antibody. Certain fully human monoclonal antibodies can be obtained from
engineered mouse strains as follows. One can engineer mouse strains
deficient in mouse antibody production with large fragments of the human
Ig loci in anticipation that such mice would produce human antibodies in
the absence of mouse antibodies. Large human Ig fragments may preserve
the large variable gene diversity as well as the proper regulation of
antibody production and expression. By exploiting the mouse machinery for
antibody diversification and selection and the lack of immunological
tolerance to human proteins, the reproduced human antibody repertoire in
these mouse strains may yield high affinity fully human antibodies
against any antigen of interest, including human antigens. Using the
hybridoma technology, antigen-specific human MAbs with the desired
specificity may be produced and selected. Certain exemplary methods are
described in WO 98/24893, U.S. Pat. No. 5,545,807, EP 546073B1, and EP
546073A1.

[0129]In certain embodiments, one may use constant regions from species
other than human along with the human variable region(s). In certain
embodiments, one may use constant regions from human along with variable
region(s) from species other than human.

Certain Exemplary Antibody Structure

[0130]Naturally occurring antibody structural units typically comprise a
tetramer. Each such tetramer typically is composed of two identical pairs
of polypeptide chains, each pair having one full-length light chain (in
certain embodiments, about 25 kDa) and one full-length heavy chain (in
certain embodiments, about 50-70 kDa).

[0131]The amino-terminal portion of each chain typically includes a
variable region (VH in the heavy chain and VL in the light
chain) of about 100 to 110 or more amino acids that typically is
responsible for antigen recognition. The carboxy-terminal portion of each
chain typically defines a constant region (CH domains in the heavy
chain and CL in the light chain) that may be responsible for
effector function. Antibody effector functions include activation of
complement and stimulation of opsonophagocytosis. Human light chains are
typically classified as kappa and lambda light chains. Heavy chains are
typically classified as mu, delta, gamma, alpha, or epsilon, and define
the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG
has several subclasses, including, but not limited to, IgG1, IgG2, IgG3,
and IgG4. IgM has subclasses including, but not limited to, IgM1 and
IgM2. IgA is similarly subdivided into subclasses including, but not
limited to, IgA1 and IgA2. Within full-length light and heavy chains,
typically, the variable and constant regions are joined by a "J" region
of about 12 or more amino acids, with the heavy chain also including a
"D" region of about 10 more amino acids. See, e.g., Fundamental
Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). The
variable regions of each light/heavy chain pair typically form the
antigen binding site.

[0132]The variable regions typically exhibit the same general structure of
relatively conserved framework regions (FR) joined by three hypervariable
regions, also called complementarity determining regions or CDRs. The
CDRs from the heavy and light chains of each pair typically are aligned
by the framework regions, which may enable binding to a specific epitope.
From N-terminal to C-terminal, both light and heavy chain variable
regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3,
and FR4. The assignment of amino acids to each domain is typically in
accordance with the definitions of Kabat Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda, Md.
(1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987);
Chothia et al. Nature 342:878-883 (1989).

[0134]In certain embodiments, functional domains, CH1, CH2,
CH3, and intervening sequences can be shuffled to create a different
antibody constant region. For example, in certain embodiments, such
hybrid constant regions can be optimized for half-life in serum, for
assembly and folding of the antibody tetramer, and/or for improved
effector function. In certain embodiments, modified antibody constant
regions may be produced by introducing single point mutations into the
amino acid sequence of the constant region and testing the resulting
antibody for improved qualities, e.g., one or more of those listed above.

[0135]In certain embodiments, an antibody of one isotype is converted to a
different isotype by isotype switching without losing its specificity for
a particular target molecule. Methods of isotype switching include, but
are not limited to, direct recombinant techniques (see e.g., U.S. Pat.
No. 4,816,397) and cell-cell fusion techniques (see e.g., U.S. Pat. No.
5,916,771), among others. In certain embodiments, an antibody can be
converted from one subclass to another subclass using techniques
described above or otherwise known in the art without losing its
specificity for a particular target molecule, including, but not limited
to, conversion from an IgG2 subclass to an IgG1, IgG3, or IgG4 subclass.

Certain Bispecific or Bifunctional Antibodies

[0136]A bispecific or bifunctional antibody typically is an artificial
hybrid antibody having two different heavy/light chain pairs and two
different binding sites. Bispecific antibodies may be produced by a
variety of methods including, but not limited to, fusion of hybridomas or
linking of Fab' fragments. See, e.g., Songsivilai & Lachmann Clin. Exp.
Immunol. 79: 315-321 (1990), Kostelny et al. J. Immunol. 148:1547-1553
(1992).

Certain Preparation of Antibodies

[0137]In certain embodiments, antibodies can be expressed in cell lines
other than hybridoma cell lines. In certain embodiments, sequences
encoding particular antibodies, including chimeric antibodies, can be
used for transformation of a suitable mammalian host cell. According to
certain embodiments, transformation can be by any known method for
introducing polynucleotides into a host cell, including, for example
packaging the polynucleotide in a virus (or into a viral vector) and
transducing a host cell with the virus or by transfecting a vector using
procedures known in the art, as exemplified by U.S. Pat. Nos. 4,399,216;
4,912,040; 4,740,461; and 4,959,455.

[0138]In certain embodiments, an expression vector comprises one or more
polynucleotide sequences discussed herein, including, but not limited to,
polynucleotide sequences encoding one or more antibodies. In certain
embodiments, a method of making a polypeptide comprising producing the
polypeptide in a cell comprising any of the above expression vectors in
conditions suitable to express the polynucleotide contained therein to
produce the polypeptide is provided.

[0139]In certain embodiments, an expression vector expresses an antibody
heavy chain. In certain embodiments, an expression vector expresses an
antibody light chain. In certain embodiments, an expression vector
expresses both an antibody heavy chain and an antibody light chain. In
certain embodiments, a method of making an antibody comprising producing
the antibody in a cell comprising at least one of expression vectors in
conditions suitable to express the polynucleotides contained therein to
produce the antibody is provided.

[0140]In certain embodiments, the transfection procedure used may depend
upon the host to be transformed. Certain methods for introduction of
heterologous polynucleotides into mammalian cells are known in the art
and include, but are not limited to, dextran-mediated transfection,
calcium phosphate precipitation, polybrene mediated transfection,
protoplast fusion, electroporation, encapsulation of the
polynucleotide(s) in liposomes, and direct microinjection of the DNA into
nuclei.

[0141]Certain mammalian cell lines available as hosts for expression are
known in the art and include, but are not limited to, many immortalized
cell lines available from the American Type Culture Collection (ATCC),
including but not limited to Chinese hamster ovary (CHO) cells, E5 cells,
HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS),
human hepatocellular carcinoma cells (e.g., Hep G2), NS0 cells, SP20
cells, Per C6 cells, 293 cells, and a number of other cell lines. In
certain embodiments, cell lines may be selected through determining which
cell lines have high expression levels and produce antibodies with
constitutive antigen binding properties.

[0142]In certain embodiments, the vectors that may be transfected into a
host cell comprise control sequences that are operably linked to a
polynucleotide encoding an antibody. In certain embodiments, control
sequences facilitate expression of the linked polynucleotide, thus
resulting in the production of the polypeptide encoded by the linked
polynucleotide. In certain embodiments, the vector also comprises
polynucleotide sequences that allow chromosome-independent replication in
the host cell. Exemplary vectors include, but are not limited to,
plasmids (e.g., BlueScript, puc, etc.), cosmids, and YACS.

Certain Expression of Recombinant Polypeptides

[0143]In certain embodiments, recombinant expression vectors are used to
amplify or express DNA encoding polypeptides, for example, including, but
not limited to TNF-R. In certain embodiments, recombinant expression
vectors are replicable DNA constructs which have synthetic or
cDNA-derived DNA fragments encoding mammalian TNF-R or bioequivalent
analogs operably linked to suitable transcriptional or translational
regulatory elements derived from mammalian, microbial, viral or insect
genes. Various recombinant expression vectors suitable for expression of
synthetic or cDNA-derived DNA fragments encoding polypeptides are well
known to one skilled in the art. Certain exemplary recombinant expression
vectors are described in U.S. Pat. No. 5,945,397.

[0144]In certain embodiments, transformed host cells are cells which have
been transformed or transfected with TNF-R vectors constructed using
recombinant DNA techniques. Transformed host cells ordinarily express
TNF-R, but host cells transformed for purposes of cloning or amplifying
TNF-R DNA do not need to express TNF-R. In certain embodiments, expressed
TNF-R will be deposited in the cell membrane or secreted into the culture
supernatant, depending on the TNF-R DNA selected. Exemplary host cells
for expression of mammalian TNF-R include, but are note limited to,
prokaryotes, yeast or higher eukaryotic cells, wherein the expression of
TNF-R is under the control of appropriate promoters. Prokaryotes include
gram negative or gram positive organisms, for example E. coli or bacilli.
Higher eukaryotic cells include, but are not limited to, established cell
lines of mammalian origin. In certain embodiments, cell-free translation
systems could also be employed to produce mammalian TNF-R using RNAs
derived from the DNA constructs containing TNF-R. Certain exemplary
cloning and expression vectors for use with bacterial, fungal, yeast, and
mammalian cellular hosts are described by Pouwels et al. (Cloning
Vectors: A Laboratory Manual, Elsevier, N.Y., 1985).

[0145]In certain embodiments, prokaryotic expression hosts are used for
expression of TNF-R. In certain embodiments, prokaryotic expression
vectors generally comprise one or more phenotypic selectable markers, for
example a gene encoding proteins conferring antibiotic resistance or
supplying an auxotrophic requirement, and an origin of replication
recognized by the host to ensure amplification within the host. Exemplary
prokaryotic hosts for transformation include E. coli, Bacillus subtilis,
Salmonella typhimurium, and various species within the genera
Pseudomonas, Streptomyces, and Staphyolococcus. Various prokaryotic
expression vectors and methods of use are well known to one skilled in
the art. Certain prokaryotic expression vectors are described in U.S.
Pat. No. 5,945,397.

[0146]In certain embodiments, recombinant TNF-R proteins are expressed in
yeast hosts, for example, Saccharomyces cerevisiae, and yeast of other
genera, such as Pichia or Kluyveromyces. Various yeast expression vectors
and methods of use are well known to one skilled in the art. Certain
exemplary yeast expression vectors and methods of use are described in R.
Hitzeman et al., European Patent Application Publication No. 0073657, and
in Sherman et al., Laboratory Course Manual for Methods in Yeast
Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1986.

[0147]In certain embodiments, mammalian or insect cell culture systems are
employed to express recombinant protein. Examples of suitable mammalian
host cell lines include, but are not limited to, the COS-7 lines of
monkey kidney cells, described by Gluzman (Cell 23:175, 1981), and other
cell lines capable of expressing an appropriate vector including, for
example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK
cell lines. Various mammalian and insect cell culture systems and methods
of use are well known to one skilled in the art. Certain such exemplary
systems are described in U.S. Pat. No. 5,945,397.

[0148]In certain embodiments, recombinant expression vectors comprising
TNF-R cDNAs are stably integrated into a host cell's DNA. In certain
embodiments, elevated levels of expression product is achieved by
selecting for cell lines having amplified numbers of vector DNA. In
certain embodiments, cell lines having amplified numbers of vector DNA
are selected, for example, by transforming a host cell with a vector
comprising a DNA sequence which encodes an enzyme which is inhibited by a
known drug. In certain embodiments, the vector also comprises a DNA
sequence which encodes the desired protein, e.g., TNF-R. In certain
embodiments, the host cell is co-transformed with a second vector which
comprises the DNA sequence encoding the desired protein, e.g., TNF-R. In
certain embodiments, the transformed or co-transformed host cells are
then cultured in increasing concentrations of the known drug, thereby
selecting for drug-resistant cells which may contain amplified copies of
the vector encoding the enzyme as well as the vector DNA encoding the
desired protein (TNF-R) in the host cell's DNA.

[0149]Certain exemplary systems for such co-amplification include, include
but are not limited to, use the gene for dihydrofolate reductase (DHFR),
which can be inhibited by the drug methotrexate (MTX); and use of the
gene for glutamine synthetase (GS), which is responsible for the
synthesis of glutamate and ammonia using the hydrolysis of ATP to ADP and
phosphate to drive the reaction. Those systems are well known to those
skilled in the art. In addition, the GS co-amplification system,
appropriate recombinant expression vectors and cells lines, are described
in the following PCT applications: WO 87/04462, WO 89101036, WO 89/10404
and WO 86/05807.

[0150]In certain embodiments, recombinant proteins are expressed by
co-amplification of DHFR or GS in a mammalian host cell, such as Chinese
Hamster Ovary (CHO) cells, or alternatively in a murine myeloma cell
line, such as SP2/0-Ag14 or NS0 or a rat myeloma cell line, such as
YB2/3.0-Ag20, disclosed in PCT applications WO/89/10404 and WO 86/05807.

[0151]Certain additional eukaryotic vectors for expression of TNF-R DNA,
including the vector, pCAV/NOT, are described in U.S. Pat. No. 5,945,397.

Purification of Recombinant TNF-R

[0152]In certain embodiments, purified mammalian TNF receptors or analogs
are prepared by culturing suitable host/vector systems to express the
recombinant translation products of the TNF-R DNAs, which are then
purified from culture media or cell extracts.

[0153]In certain embodiments, supernatants from systems which secrete
recombinant protein into culture media are first concentrated using a
commercially available protein concentration filter, for example, an
Amicon or Millipore Pellicon ultrafiltration unit. In certain
embodiments, following the concentration step, the concentrate is applied
to a suitable purification matrix. Exemplary purification matrices
include, but are not limited to, a TNF, lectin or antibody polypeptide
bound to a suitable support; an anion exchange resin comprising, for
example, pendant diethylaminoethyl (DEAE) groups, wherein the matrix is
acrylamide, agarose, dextran, cellulose or other types commonly employed
in protein purification; a cation exchange resin, comprising various
insoluble matrices comprising sulfopropyl or carboxymethyl groups.

[0154]In certain embodiments, one or more reversed-phase high performance
liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC
media, e.g., silica gel having pendant methyl or other aliphatic groups,
are employed to further purify a TNF-R composition. In certain
embodiments, some or all of the foregoing purification steps, in various
combinations, are employed to provide a homogeneous recombinant protein.

[0155]In certain embodiments, recombinant protein produced in bacterial
culture is typically isolated by initial extraction from cell pellets,
followed by one or more concentration, salting-out, aqueous ion exchange
or size exclusion chromatography steps. In certain embodiments, high
performance liquid chromatography (HPLC) is employed for final
purification steps. In certain embodiments, microbial cells employed in
expression of recombinant mammalian TNF-R are disrupted by any convenient
method, for example, freeze-thaw cycling, sonication, mechanical
disruption, or use of cell lysing agents.

[0157]In certain embodiments, a composition comprising at least one
specific binding agent, at least one stabilizing agent, and a buffering
agent is provided. In certain such embodiments, the composition further
comprises at least one additional pharmaceutical agent. In certain
embodiments, the specific binding agent is a specific binding agent to
RANKL, a specific binding agent to TNF, and/or a specific binding agent
to IL-1R1. In certain embodiments, the specific binding agent is a
specific binding agent to RANKL, wherein the specific binding agent to
RANKL is an antibody which specifically binds RANKL. In certain
embodiments, the antibody is αRANKL-1. In certain embodiments, the
specific binding agent is a specific binding agent to TNF, wherein the
specific binding agent to TNF is a soluble TNF receptor. In certain
embodiments, the soluble TNF receptor is sTNFR:Fc. In certain
embodiments, the specific binding agent is a specific binding agent to
IL-1R1, wherein the specific binding agent is an antibody which
specifically binds IL-1R1. In certain embodiments, the antibody is
selected from 15C4, 26F5 and 27F2 as described in U.S. Publication No.
2004/0097712.

[0158]In certain embodiments, the at least one specific binding agent to
RANKL is at a concentration of 1 mg/ml to 150 mg/ml. In certain such
embodiments, the at least one specific binding agent to RANKL is an
antibody which specifically binds RANKL. In certain embodiments, the
antibody is αRANKL-1. Certain exemplary concentrations of the at
least one specific binding agent to RANKL include, but are not limited
to, 30 mg/ml, 60 mg/ml, 70 mg/ml, 105 mg/ml, and 120 mg/ml. In certain
embodiments, compositions will include more than one different specific
binding agent to RANKL. In certain such embodiments, the more than one
specific binding agents to RANKL bind more than one epitope.

[0159]In certain embodiments, the at least one specific binding agent to
TNF is at a concentration of 1 mg/ml to 150 mg/ml. In certain such
embodiments, the at least one specific binding agent to TNF is present at
a concentration of 50 mg/ml. In certain embodiments, compositions will
include more than one different specific binding agent to TNF. In certain
such embodiments, the more than one specific binding agents to TNF bind
more than one epitope. Exemplary formulations for a specific binding
agent to TNF, including soluble TNFR:Fc, can be found in U.S. Patent
Publication No. 2007-0243185, incorporated herein by reference in its
entirety.

[0160]In certain embodiments, the at least one specific binding agent to
IL-1R1 is at a concentration of 1 mg/ml to 200 mg/ml. In certain such
embodiments, the at least one specific binding agent to IL-1R1 is an
antibody which specifically binds IL-1R1. In certain embodiments, the
antibody is selected from 15C4, 26F5 or 27F2 as described in U.S.
Publication No. 2004/0097712. Certain exemplary concentrations of the at
least one specific binding agent to IL-1R1 include, but are not limited
to, 30 mg/ml, 70 mg/ml, 100 mg/ml, and 150 mg/ml. In certain embodiments,
compositions will include more than one different specific binding agent
to IL-1R1. In certain such embodiments, the more than one specific
binding agents to IL-1R1 bind more than one epitope.

[0161]In certain embodiments, the pH of a composition comprising a
buffering agent is below 6.6. In certain embodiments, the pH of a
composition comprising a buffering agent is between 5.5 and 6.5. In
certain such embodiments, the pH is 6.3. In certain embodiments, the pH
of a composition comprising a buffering agent is between 4.5 and 5.5. In
certain such embodiments, the pH is 5.2. Exemplary buffering agents
include, but are not limited to, acetate, histidine, phosphate,
glutamate, and propionate. In certain embodiments, the concentration of a
buffering agent ranges from 1 mM to 50 mM. In certain embodiments, the
concentration of the buffering agent is 25 mM. In certain embodiments,
the concentration of the buffering agent is 10 mM.

[0162]In certain embodiments, the composition further comprises at least
one sugar. As used herein, the term "sugar" refers to monosaccharides
such as glucose and mannose, or polysaccharides including disaccharides
such as sucrose and lactose, as well as sugar derivatives including sugar
alcohols and sugar acids. Sugar alcohols include, but are not limited to,
mannitol, xylitol, erythritol, threitol, sorbitol and glycerol. A
non-limiting example of a sugar acid is L-gluconate. Certain exemplary
sugars include, but are not limited to, trehalose and glycine. In certain
embodiments, a sugar is provided at a concentration between 0.5% and
9.5%. In certain embodiments, a sugar is 1% sucrose. In certain
embodiments, a sugar is 5.0% sorbitol.

[0163]In certain embodiments, the composition further comprises at least
one surfactant. As used herein, the term "surfactant" refers to a
surface-active agent comprising a hydrophobic portion and a hydrophilic
portion. Examples of surfactants include, but are not limited to,
detergents and bile acid salts. In certain instances, surfactants are
categorized as anionic, nonionic, zwitterionic, or cationic, depending on
whether they comprise one or more charged group. Nonionic surfactants
contain non-charged polar groups and have no charge. Certain exemplary
nonionic surfactants include, but are not limited to, polyethylene glycol
(PEG), including, but not limited to, PEG 8000, and polysorbate,
including but not limited to, polysorbate 80 (Tween® 80) and
polysorbate 20 (Tween® 20), Triton X-100,
polyoxypropylene-polyoxyethylene esters (Pluronic®), and NP-40. In
certain embodiments, the surfactant is provided at a concentration
between 0.001% and 1.0%. In certain embodiments, the surfactant is
provided at a concentration between 0.003% and 0.3%. In certain
embodiments, the surfactant is provided at a concentration of 0.01%. In
certain embodiments, the surfactant is provided at a level below the
critical micelle concentration (CMC) of the surfactant. In certain such
embodiments, the composition comprises a human monoclonal antibody to
human IL-1R1 and polysorbate 20, which has a CMC of 0.007%, and the
concentration of polysorbate 20 is 0.004%. In certain embodiments, the
surfactant is provided at a level above the CMC of the surfactant. In
certain such embodiments, the composition comprises αRANKL-1 and
polysorbate 20, which has a CMC of 0.007%, and the concentration of
polysorbate 20 is 0.01%.

[0164]In certain embodiments, a composition comprising at least one
specific binding agent, at least one stabilizing agent, and a buffering
agent provides stabilization of at least one specific binding agent. In
certain embodiments, the specific binding is a specific binding agent to
RANKL, a specific binding agent to TNF, and/or a specific binding agent
to IL-1R1. In certain embodiments, the specific binding agent is a
specific binding agent to RANKL, wherein the specific binding agent to
RANKL is an antibody which specifically binds RANKL. In certain
embodiments, the antibody is αRANKL-1. In certain embodiments, the
specific binding agent is a specific binding agent to TNF, wherein the
specific binding agent to TNF is a soluble TNF receptor. In certain
embodiments, the soluble TNF receptor is sTNFR:Fc. In certain
embodiments, the specific binding agent is a specific binding agent to
IL-1R1, wherein the specific binding agent to IL-1R1 is an antibody which
specifically binds IL-1R1. In certain embodiments, the antibody is
selected from 15C4, 26F5 and 27F2 as described in U.S. Publication No.
2004/0097712. In certain embodiments, the composition provides
stabilization with respect to formation of fewer aggregates and/or
dimers. In certain embodiments, the composition provides stabilization
with respect to formation of fewer chemically altered forms.

[0165]In certain embodiments, the presence and degree of aggregation
and/or chemically altered forms of a particular protein molecule in a
sample can be determined by suitable methods known in the art, such as
size exclusion chromatography (SEC), for example, also known as gel
filtration chromatography or molecular sieving chromatography. In certain
embodiments, a suitable method for determining the presence of aggregates
and/or chemically altered forms in a sample is gel electrophoresis under
non-denaturing conditions. The "gel" refers to a matrix of water and a
polymer such as agarose or polymerized acrylamide. These methods separate
molecules on the basis of the size of the molecule compared to the size
of the pores of the gel. Certain other methods of measuring aggregation
and/or chemically altered forms include, but are not limited to,
hydrophobic interaction chromatography (HIC) and high performance liquid
chromatography (HPLC). HPLC provides a separation based on any one of
adsorption, ion exchange, size exclusion, HIC, or reverse phase
chromatography. HIC separates native proteins on the basis of their
surface hydrophobicity between the hydrophobic moieties of the protein
and insoluble, immobilized hydrophobic groups on the matrix. Generally,
the protein preparation in a high salt buffer is loaded on the HIC
column. The salt in the buffer interacts with water molecules to reduce
the solvation of the proteins in solution, thereby exposing hydrophobic
regions in the protein which are then adsorbed by the hydrophobic groups
on the matrix. The more hydrophobic the molecule, the less salt is needed
to promote binding. Usually, a decreasing salt gradient is used to elute
proteins from a column. As the ionic strength decreases, the exposure of
the hydrophilic regions of the protein increases and proteins elute from
the column in order of increasing hydrophobicity. See, for example,
Protein Purification, 2d Ed., Springer-Verlag, New York, 176-179 (1988).
In certain embodiments, the separations are improved through the use of
high-resolution columns and decreased column retention times. See, for
example, Chicz et al., Methods in Enzymology 182, pp. 392-421 (1990).
Additional exemplary methods for monitoring protein stability are found
in Lee, V., ed. Peptide and Protein Drug Delivery (Marcel Dekker, Inc.,
New York, N.Y., 1991). In certain embodiments, protein stability is
measured at a certain temperature for a certain period of time. In
certain embodiments, a specific binding agent to RANKL, a specific
binding agent to TNF, and/or a specific binding agent to IL-1R1 is
stabilized in a composition stored at room temperature (between
21° C. and 29° C.). Exemplary storage times include, but
are not limited to, at least 1 month, at least 3 months, at least 6
months, at least 9 months, at least 12 months, at least 18 months, and at
least 24 months. In certain embodiments, a specific binding agent to
RANKL, a specific binding agent to TNF, and/or a specific binding agent
to IL-1R1 is stabilized in a composition stored between 2° C. and
8° C. Exemplary storage time include, but are not limited to, at
least 6 months, at least 9 months, at least 12 months, at least 18
months, and at least 24 months.

[0166]In certain embodiments, a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1
is prepared, purified, and formulated as a liquid pharmaceutical
composition. In certain embodiments, after preparation and purification,
a specific binding agent to RANKL, a specific binding agent to TNF,
and/or a specific binding agent to IL-1R1 is stored prior to formulation.
In certain such embodiments, the specific binding agent to RANKL, the
specific binding agent to TNF, and/or the specific binding agent to
IL-1R1 is frozen, for example, at -20° C. or lower. In certain
such embodiments, the specific binding agent to RANKL, the specific
binding agent to TNF, and/or the specific binding agent to IL-1R1 is
thawed at room temperature for further formulation. In certain
embodiments, a liquid pharmaceutical formulation comprises a
therapeutically effective amount a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1.
In certain embodiments, the amount of specific binding agent to RANKL,
specific binding agent to TNF, and/or specific binding agent to IL-1R1 to
formulate in a formulation will be determined by one skilled in the art,
depending upon, for example, the route of administration and desired dose
volume. In certain embodiments, the pharmaceutical formulation comprises
a specific binding agent to RANKL at a concentration of 1 mg/ml to 150
mg/ml. In certain such embodiments, the specific binding agent to RANKL
is an antibody which specifically binds RANKL. In certain embodiments,
the antibody is αRANKL-1. In certain embodiments, the
pharmaceutical formulation comprises a specific binding agent to TNF at a
concentration of 1 mg/ml to 150 mg/ml. In certain such embodiments, the
specific binding agent to TNF is a soluble TNF receptor. In certain
embodiments, the soluble TNF receptor is sTNFR:Fc. In certain
embodiments, the pharmaceutical formulation comprises a specific binding
agent to IL-1R1 at a concentration of 1 mg/ml to 200 mg/ml. In certain
such embodiments, the specific binding agent to IL-1R1 is an antibody
which specifically binds IL-1R1. In certain embodiments, the antibody is
selected from 15C4, 26F5, and 27F2 as described in U.S. Publication No.
2004/0097712. In certain embodiments, a pharmaceutical formulation
comprises a therapeutically effective amount a specific binding agent to
RANKL and a buffer that maintains the pH of the formulation below 6.6. In
certain embodiments, a buffer maintains the pH of the formulation between
4.5 and 5.5. In certain such embodiments, a buffer maintains the pH of
the formulation at 5.2. In certain embodiments, a pharmaceutical
formulation comprises a therapeutically effective amount a specific
binding agent to IL-1R1 and a buffer that maintains the pH of the
formulation below 6.6. In certain embodiments, a buffer maintains the pH
of the formulation between 4.5 and 5.5. In certain such embodiments, a
buffer maintains the pH of the formulation at 5.0. In certain
embodiments, a pharmaceutical formulation comprises a therapeutically
effective amount a specific binding agent to TNF and a buffer that
maintains the pH of the formulation between 5.5 and 6.5. In certain
embodiments, a buffer maintains the pH of the formulation at 6.3.

[0167]In certain embodiments, specific binding agents including, but not
limited to, antibodies and soluble polypeptides, which bind to a
particular protein and block interaction with other binding compounds may
have therapeutic use. In this application, when discussing the use of
antibodies and soluble polypeptides to treat diseases or conditions, such
use may include use of compositions comprising antibodies or soluble
polypeptides; and/or combination therapies comprising antibodies or
soluble polypeptides and one or more additional active ingredients. When
antibodies or soluble polypeptides are used to "treat" a disease or
condition, such treatment may or may not include prevention of the
disease or condition.

[0168]In certain embodiments, a specific binding agent including, but not
limited to, an antibody or a soluble polypeptide, is administered alone.
In certain embodiments, an antibody or soluble polypeptide is
administered prior to the administration of at least one other
therapeutic agent. In certain embodiments, an antibody or soluble
polypeptide is administered concurrent with the administration of at
least one other therapeutic agent. In certain embodiments, an antibody or
soluble polypeptide is administered subsequent to the administration of
at least one other therapeutic agent. Exemplary therapeutic agents,
include, but are not limited to, at least one cancer therapy agent.
Exemplary cancer therapy agents include, but are not limited to,
radiation therapy and chemotherapy.

[0169]In certain embodiments, pharmaceutical compositions comprising
specific binding agents, e.g., antibodies or soluble polypeptides, can be
administered in combination therapy, i.e., combined with other agents.
Exemplary agents include, but are not limited to, in vitro synthetically
prepared chemical compositions, antibodies, antigen binding regions,
radionuclides, and combinations and conjugates thereof. In certain
embodiments, an agent may act as an agonist, antagonist, allosteric
modulator, or toxin. In certain embodiments, an agent may act to inhibit
or stimulate its target (e.g., receptor or enzyme activation or
inhibition), and thereby promote cell death or arrest cell growth. In
certain embodiments, the combination therapy comprises a specific binding
agent to RANKL, a specific binding agent to TNF, and/or a specific
binding agent to IL-1R1, in combination with at least one anti-angiogenic
agent. In certain embodiments, the specific binding agent to RANKL is an
antibody which specifically binds RANKL. In certain embodiments, the
antibody is αRANKL-1. In certain embodiments, the specific binding
agent to TNF is a soluble TNF receptor. In certain embodiments, the
soluble TNF receptor is sTNFR:Fc. In certain embodiments, the specific
binding agent to IL-1R1 is an antibody which specifically binds IL-1R1.
In certain embodiments, the antibody is selected from 15C4, 26F5 and 27F2
as described in U.S. Publication No. 2004/0097712.

[0171]Exemplary cancer therapies, which may be administered with a
specific binding agent to RANKL, a specific binding agent to TNF, and/or
a specific binding agent to IL-1R1, also include, but are not limited to,
targeted therapies. Examples of targeted therapies include, but are not
limited to, use of therapeutic antibodies. Exemplary therapeutic
antibodies, include, but are not limited to, mouse, mouse-human chimeric,
CDR-grafted, humanized and fully human antibodies, and synthetic
antibodies, including, but not limited to, those selected by screening
antibody libraries. Exemplary antibodies include, but are not limited to,
those which bind to cell surface proteins Her2, CDC20, CDC33, mucin-like
glycoprotein, VEGF, and epidermal growth factor receptor (EGFR) present
on tumor cells, and optionally induce a cytostatic and/or cytotoxic
effect on tumor cells displaying these proteins.

[0173]In certain embodiments, a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1
may be administered prior to, concurrent with, and subsequent to
treatment with a cancer therapy agent. In certain embodiments, a specific
binding agent to RANKL, a specific binding agent to TNF, and/or a
specific binding agent to IL-1R1 may be administered prophylactically to
prevent or mitigate the onset of bone loss by metastatic cancer. In
certain embodiments, a specific binding agent to RANKL, a specific
binding agent to TNF, and/or a specific binding agent to IL-1R1 may be
administered for the treatment of an existing condition of bone loss due
to metastasis.

[0175]In certain embodiments, a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1
may be used alone or with at least one additional therapeutic agent for
the treatment of cancer. In certain embodiments, a specific binding agent
to RANKL, a specific binding agent to TNF, and/or a specific binding
agent to IL-1R1 is used in conjunction with a therapeutically effective
amount of an additional therapeutic agent.

[0176]In certain embodiments, a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1
is used with one or more particular therapeutic agents to treat various
cancers. In certain embodiments, a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1
is used with one or more particular therapeutic agents to treat or
prevent malaria. In certain embodiments, a specific binding agent to
RANKL, a specific binding agent to TNF, and/or a specific binding agent
to IL-1R1 is used with one or more particular therapeutic agents to treat
or prevent proliferative diabetic retinopathy. In certain embodiments, in
view of the condition and the desired level of treatment, two, three, or
more agents may be administered. In certain embodiments, such agents may
be provided together by inclusion in the same formulation. In certain
embodiments, such agents and a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1
may be provided together by inclusion in the same formulation. In certain
embodiments, such agents may be formulated separately and provided
together by inclusion in a treatment kit. In certain embodiments, such
agents and a specific binding agent to RANKL, a specific binding agent to
TNF, and/or a specific binding agent to IL-1R1 may be formulated
separately and provided together by inclusion in a treatment kit. In
certain embodiments, such agents may be provided separately. In certain
embodiments, when administered by gene therapy, the genes encoding
protein agents and/or a specific binding agent to RANKL, a specific
binding agent to TNF, and/or a specific binding agent to IL-1R1 may be
included in the same vector. In certain embodiments, the genes encoding
protein agents and/or a specific binding agent to RANKL, a specific
binding agent to TNF, and/or a specific binding agent to IL-1R1 may be
under the control of the same promoter region. In certain embodiments,
the genes encoding protein agents and/or a specific binding agent to
RANKL, a specific binding agent to TNF, and/or a specific binding agent
to IL-1R1 may be in separate vectors.

[0177]It is understood that the response by individual patients to the
aforementioned medications or combination therapies may vary, and an
appropriate efficacious combination of drugs for each patient may be
determined by his or her physician.

[0180]In certain embodiments, therapies comprising a specific binding
agent to RANKL, a specific binding agent to TNF, and/or a specific
binding agent to IL-1R1 and at least one serine protease inhibitor, and
methods of treatment using such therapies are provided. In certain
embodiments, a therapy comprises a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1,
a serine protease inhibitor, and at least one additional agent described
herein.

[0181]In certain instances, a disturbance of the protease/protease
inhibitor balance can lead to protease-mediated tissue destruction,
including, but not limited to, tumor invasion of normal tissue leading to
metastasis.

[0182]In certain embodiments, a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1
may be used with at least one therapeutic agent for inflammation. In
certain embodiments, a specific binding agent to RANKL, a specific
binding agent to TNF, and/or a specific binding agent to IL-1R1 may be
used with at least one therapeutic agent for an immune disorder. Certain
exemplary therapeutic agents for inflammation are described, e.g., in C.
A. Dinarello and L. L. Moldawer Proinflammatory and Anti-Inflammatory
Cytokines in Rheumatoid Arthritis: A Primer for Clinicians Third Edition
(2001) Amgen Inc. Thousand Oaks, Calif.

[0183]In certain embodiments, pharmaceutical compositions include more
than one different specific binding agent to RANKL, specific binding
agent to TNF, and/or specific binding agent to IL-1R1. In certain such
embodiments, the more than one specific binding agents to RANKL bind more
than one epitope. In certain such embodiments, the more than one specific
binding agents to TNF bind more than one epitope. In certain such
embodiments, the more than one specific binding agents to IL-1R1 bind
more than one epitope.

[0184]In certain embodiments, liquid compositions comprising one or more
specific binding agent to RANKL, one or more specific binding agent to
TNF, and/or one or more specific binding agent to IL-1R1 are prepared as
aqueous or nonaqueous solutions or suspensions for subsequent
administration to a patient.

[0185]In certain embodiments, materials for compositions are nontoxic to
recipients at the dosages and concentrations employed.

[0187]In certain embodiments, a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1
is linked to a half-life extending vehicle known in the art. Such
vehicles include, but are not limited to, the Fc domain, polyethylene
glycol (PEG), polyoxyethylated polyols, and dextran. Such vehicles and
methods are described, e.g., in U.S. Pat. Nos. 4,179,337; 4,495,285;
4,609,546; 4,766,106; 6,660,843; and published PCT Application No. WO
99/25044. In certain instances, PEG is soluble in water at room
temperature and has the general formula:
R(O--CH2--CH2)nO--R where R is hydrogen, or a protective
group, including, but not limited to, an alkyl or alkanol group, and
where "n" is a positive integer. In certain embodiments, the protective
group has between 1 and 8 carbons. In certain such embodiments, the
protective group is methyl. In certain embodiments, "n" is between 1 and
1,000. In certain embodiments, PEG has an average molecular weight
between 1,000 and 40,000. Those ranges and any ranges discussed in this
application include the endpoints and all values between the endpoints.
In certain embodiments, PEG has at least one hydroxy group. In certain
such embodiments, the hydroxy group is a terminal hydroxy group. In
certain such embodiments, the terminal hydroxy group is activated by
N-hydroxysuccinimide to react with a free amino group on a specific
binding agent to RANKL, a specific binding agent to TNF, and/or a
specific binding agent to IL-1R1 to form a covalently conjugated
molecule. In certain embodiments, the type and amount of the reactive
groups may be varied to achieve a covalently conjugated PEG/specific
binding agent. Preparation of conjugated PEG molecules is within the
skill of the art.

[0188]In certain embodiments, a half-life extending vehicle is
polyoxyethylated polyol. Exemplary polyoxyethylated polyols include, but
are not limited to, polyoxyethylated sorbitol, polyoxyethylated glucose,
and polyoxyethylated glycerol (POG). In certain embodiments, POG has an
average molecular weight between 1,000 and 40,000. That range and any
ranges discussed in this application include the endpoints and all values
between the endpoints. Certain exemplary structures of POG are found, for
example, in Knauf et al., J. Biol. Chem. 263:15064-15070 (1988). Certain
exemplary POG conjugates are found, for example, in U.S. Pat. No.
4,766,106.

[0189]In certain embodiments, the optimal pharmaceutical composition will
be determined by one skilled in the art depending upon, for example, the
intended route of administration, delivery format and desired dosage.
See, for example, Remington's Pharmaceutical Sciences, supra. In certain
embodiments, such compositions may influence the physical state,
stability, rate of in vivo release and rate of in vivo clearance of the
antibodies of the invention.

[0190]In certain embodiments, the primary vehicle or carrier in a
pharmaceutical composition is aqueous in nature. For example, in certain
embodiments, a suitable vehicle or carrier may be water for injection,
physiological saline solution or artificial cerebrospinal fluid, possibly
supplemented with other materials common in compositions for parenteral
administration. In certain embodiments, the vehicle or carrier is
sterile. In certain embodiments, additional components are included.
Exemplary additional components include, but are not limited to, fixed
oils; polyethylene glycols; glycerin; propylene glycol and other
synthetic solvents; antibacterial agents including, but not limited to,
benzyl alcohol and methyl parabens; antioxidants including, but not
limited to, ascorbic acid and sodium bisulfite; and chelating agents
including, but not limited to ethylenediaminetetraacetic acid. In certain
embodiments, neutral buffered saline or saline mixed with serum albumin
are further exemplary vehicles. In certain embodiments, pharmaceutical
compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer
of about pH 5.0-5.5, or glutamate buffer of about pH 5.0-5.5, or
succinate buffer of about pH 5.0-5.5, or histidine buffer of about pH
5.0-5.5, or aspartate buffer of about pH 5.0-5.5, or phosphate buffer of
about pH 6.0-6.5, which may further include sucrose, sorbitol or a
suitable substitute therefore. In certain embodiments, pharmaceutical
compositions are self-buffering. See, e.g., International Application
No.: PCT/US2006/022599, published on Dec. 28, 2006. In certain
embodiments, a composition comprising a specific binding agent to RANKL,
a specific binding agent to TNF, and/or a specific binding agent to
IL-1R1, with or without at least one additional therapeutic agents, may
be prepared for storage by mixing the selected composition having the
desired degree of purity with optional formulation agents (Remington's
Pharmaceutical Sciences, supra) in the form of an aqueous solution. In
certain embodiments, a pharmaceutical composition is enclosed in a
container. Exemplary containers include, but are not limited to, an
ampoule, disposable syringe, including, but not limited to, disposable
syringe suitable for prefilling, and multiple dose vial made of glass or
plastic. In certain embodiments, a composition comprising a specific
binding agent to RANKL, a specific binding agent to TNF, and/or a
specific binding agent to IL-1R1 is contained in a prefilled syringe. In
certain embodiments, the specific binding agent to RANKL is an antibody
which specifically binds RANKL. In certain embodiments, the antibody is
αRANKL-1. In certain embodiments, the specific binding agent to TNF
is a soluble TNF receptor. In certain embodiments, the soluble TNF
receptor is sTNFR:Fc. In certain embodiments, the specific binding agent
to IL-1R1 is an antibody which specifically binds IL-1R1. In certain
embodiments, the antibody is selected from 15C4, 26F5 and 27F2 as
described in U.S. Publication No. 2004/0097712. Exemplary syringes
suitable for prefilling are described, for example, in U.S. Pat. No.
5,607,400. Syringes suitable for prefilling are available commercially
from various sources, for example, Daikyo Seiko, Ltd (Tokyo, Japan),
Becton-Dickinson (Franklin Lakes, N.J.), Bunder Glass (Dusseldorf,
Germany), and Schott-Form a Vitrum (Lebanon, Pa.).

[0191]In certain embodiments, pharmaceutical compositions can be selected
for parenteral delivery. Exemplary parenteral delivery includes, but is
not limited to, intravenous, intramuscular, intradermal, or subcutaneous
administration. In certain embodiments, the compositions may be selected
for delivery through the digestive tract, such as orally. The preparation
of such pharmaceutically acceptable compositions is within the skill of
the art.

[0192]In certain embodiments, the formulation components are present in
concentrations that are acceptable to the site of administration. In
certain embodiments, a pharmaceutical composition comprises a
therapeutically effective amount a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1
and a buffer. In certain embodiments, the specific binding agent to RANKL
is an antibody which specifically binds RANKL. In certain embodiments,
the antibody is αRANKL-1. In certain embodiments, the specific
binding agent to TNF is a soluble TNF receptor. In certain embodiments,
the soluble TNF receptor is sTNFR:Fc. In certain embodiments, the
specific binding agent to IL-1R1 is an antibody which specifically binds
IL-1R1. In certain embodiments, the antibody is selected from 15C4, 26F5
and 27F2 as described in U.S. Publication No. 2004/0097712. In certain
embodiments, buffers are used to maintain the composition at
physiological pH or at a slightly lower pH. In certain embodiments,
buffers are between pH 5.5 and pH 8.0. In certain embodiments, buffers
are between pH 5.5 and pH 6.5. In certain embodiments, buffers are
between pH 4.5 and pH 5.5. Exemplary buffers include, but are not limited
to, acids and/or salts thereof, including, but not limited to, succinic
acid or succinate, citric acid or citrate, acetic acid or acetate,
tartaric acid or tartarate, phosphoric acid or phosphate, propionic acid
or propionate, gluconic acid or gluconate, glutamic acid or glutamate,
histidine, glycine, aspartic acid or aspartate, maleic acid or maleate,
and malic acid or malate buffers. In certain instances, a "salt" refers
to an electrically-neutral substance formed between an anion of an acid
and an oppositely charged ion. In certain such instances, the oppositely
charged ion is referred to as a "counterion." Exemplary counterions
include, but are not limited to, sodium, potassium, ammonium, calcium,
and magnesium. In certain embodiments, the concentration of buffer in a
formulation is between 1 mM and 50 mM. In certain embodiments, the
concentration of buffer in a formulation is between 5 mM and 30 mM. In
certain embodiments, the concentration of buffer in a formulation is
between 10 mM and 25 mM. Those ranges and any ranges discussed in this
application include the endpoints and all values between the endpoints.
In certain embodiments, the concentration of buffer in a formulation is
10 mM. In certain embodiments, the concentration of buffer in a
formulation is 25 mM.

[0193]In certain embodiments, the pharmaceutical formulation comprises a
specific binding agent to RANKL, a specific binding agent to TNF, and/or
a specific binding agent to IL-1R1 at a concentration of 1 mg/ml to 200
mg/ml and a buffer. In certain embodiments, the buffer is at a
concentration between 1 mM and 50 mM, and the pH of the formulation is
below 6.6. In certain such embodiments, the pharmaceutical formulation
comprises a specific binding agent to RANKL at a concentration of 60
mg/ml and a buffer at a concentration of 10 mM, and the pH of the
formulation is 5.2. In certain embodiments, the specific binding agent to
RANKL is an antibody which specifically binds RANKL. In certain
embodiments, the antibody is αRANKL-1. In certain such embodiments,
the pharmaceutical formulation comprises a specific binding agent to TNF
at a concentration of 50 mg/ml and a buffer at a concentration of 25 mM,
and the pH of the formulation is 6.3. In certain embodiments, the
specific binding agent to TNF is a soluble TNF receptor. In certain
embodiments, the soluble TNF receptor is sTNFR:Fc.

[0194]In certain embodiments, a pharmaceutical formulation comprises a
therapeutically effective amount a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1
and a buffer. In certain embodiments, the buffer is a phosphate buffer or
an acetate buffer, at a concentration that maintains the pH of the
formulation below 6.6. In certain embodiments, the pH of the formulation
is between 4.0 and 6.0. The term "phosphate buffer" refers to a buffer
comprising a salt of phosphoric acid. The term "acetate buffer" refers to
a buffer comprising a salt of acetic acid. In certain embodiments, the
phosphate or acetate counterion is sodium. In certain such embodiments,
the buffer is sodium phosphate or sodium acetate. Other exemplary
counterions include, but are not limited to, potassium, ammonium,
calcium, and magnesium. In certain embodiments, the concentration of the
phosphate buffer or acetate buffer in the formulation is between 1 mM and
50 mM. In certain embodiments, the concentration of the phosphate buffer
or acetate buffer in the formulation is between 5 mM and 30 mM. In
certain embodiments, the concentration of the phosphate buffer or acetate
buffer in the formulation is between 10 mM and 25 mM. Those ranges and
any ranges discussed in this application include the endpoints and all
values between the endpoints. In certain embodiments, the concentration
of the phosphate buffer or acetate buffer in the formulation is 10 mM. In
certain embodiments, the concentration of the phosphate buffer or acetate
buffer in the formulation is 25 mM. In certain embodiments, the
pharmaceutical formulation comprises a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1
at a concentration of 1 mg/ml to 200 mg/ml and a buffer. In certain
embodiments, the buffer is a phosphate buffer or an acetate buffer, at a
concentration between 1 mM and 50 mM, and the pH of the formulation is
below 6.6. In certain such embodiments, the pharmaceutical formulation
comprises a specific binding agent to RANKL at a concentration of 60
mg/ml and acetate buffer at a concentration of 10 mM, and the pH of the
formulation is 5.2. In certain embodiments, the specific binding agent to
RANKL is an antibody which specifically binds RANKL. In certain
embodiments, the antibody is αRANKL-1. In certain embodiments, the
pharmaceutical formulation comprises a specific binding agent to TNF at a
concentration of 50 mg/ml and phosphate buffer at a concentration of 25
mM, and the pH of the formulation is 6.3. In certain such embodiments,
the specific binding agent to TNF is a soluble TNF receptor. In certain
embodiments, the soluble TNF receptor is sTNFR:Fc.

[0195]In certain embodiments, a pharmaceutical formulation comprises a
therapeutically effective amount of a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1;
a buffer at a concentration that maintains the pH of the formulation
below 6.6; and an amount of an isotonizing agent sufficient to provide a
formulation that is isotonic. In certain embodiments, the buffer is a
phosphate buffer or an acetate buffer. A formulation that is "isotonic"
has an osmolarity between 270 mOsm and 370 mOsm. In certain embodiments,
the pH of the formulation is between 4.0 and 6.0. Certain methods of
determining the isotonicity of a solution are within the knowledge of
those skilled in the art. See, e.g., Setnikar et al., J. Am. Pharm.
Assoc. 48:628-30 (1959). Exemplary isotonizing agents include, but are
not limited to, sodium chloride; amino acids, including, but not limited
to, alanine, arginine, valine, and glycine; sugars and sugar alcohols
(polyols), including, but not limited to, glucose, dextrose, fructose,
sucrose, maltose, mannitol, trehalose, glycerol, sorbitol, and xylitol;
acetic acid, other organic acids or their salts, and relatively minor
amounts of citrates or phosphates. In certain embodiments, the
isotonizing agent is provided at a concentration of at least 5%. In
certain embodiments, the isotonizing agent is sucrose at a concentration
of 9%.

[0196]In certain embodiments, a pharmaceutical formulation comprises a
therapeutically effective amount a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1;
a buffer at a concentration that maintains the pH of the formulation
below 6.6; and a surfactant. In certain embodiments, the buffer is a
phosphate buffer or an acetate buffer. In certain embodiments, the pH of
the formulation is between 4.0 and 6.0. In certain embodiments, the
surfactant is a nonionic surfactant. Certain exemplary nonionic
surfactants include, but are not limited to, polyoxyethylene sorbital
esters (polysorbates), polyoxypropylene-polyoxyethylene esters
(Pluronic®), polyoxyethylene alcohols, simethicone, polyethylene
glycols, lysophosphatidylcholine, and polyoxyethylene-p-t-octylphenols.
Certain exemplary surfactants include, but are not limited to, PEG 8000,
polysorbate 80 (Tween® 80), and polysorbate 20 (Tween® 20). In
certain embodiments, the surfactant is provided at a concentration
between 0.001% and 1.0%. In certain embodiments, the surfactant is
provided at a concentration between 0.003% and 0.3%. In certain
embodiments, the surfactant is provided at a concentration of 0.01%.
Those ranges and any ranges discussed in this application include the
endpoints and all values between the endpoints.

[0197]In certain embodiments, when parenteral administration is
contemplated, a therapeutic composition may be in the form of a
pyrogen-free, parenterally acceptable aqueous solution comprising a
desired specific binding agent to RANKL, desired specific binding agent
to TNF, and/or desired specific binding agent to IL-1R1, with or without
additional therapeutic agents, in a pharmaceutically acceptable vehicle.
In certain embodiments, a vehicle for parenteral injection is sterile
distilled water in which a specific binding agent to RANKL, a specific
binding agent to TNF, and/or a specific binding agent to IL-1R1, with or
without at least one additional therapeutic agent, is formulated as a
sterile, isotonic solution, properly preserved. In certain embodiments,
the preparation can involve the formulation of the desired molecule with
an agent, such as injectable microspheres, bio-erodible particles,
polymeric compounds (such as polylactic acid or polyglycolic acid), beads
or liposomes, that may provide for the controlled or sustained release of
the product which may then be delivered via a depot injection. In certain
embodiments, hyaluronic acid may also be used, and may have the effect of
promoting sustained duration in the circulation. In certain embodiments,
implantable drug delivery devices may be used to introduce the desired
molecule.

[0199]The pharmaceutical composition to be used for in vivo administration
typically is sterile. In certain embodiments, this may be accomplished by
filtration through sterile filtration membranes. In certain embodiments,
parenteral compositions generally are placed into a container having a
sterile access port, for example, an intravenous solution bag or vial
having a stopper pierceable by a hypodermic injection needle. In certain
embodiments, parenteral compositions are placed in a syringe suitable for
prefilling with the compositions.

[0200]In certain embodiments, the effective amount of a pharmaceutical
composition comprising a specific binding agent to RANKL, a specific
binding agent to TNF, and/or a specific binding agent to IL-1R1, with or
without at least one additional therapeutic agent, to be employed
therapeutically will depend, for example, upon the therapeutic context
and objectives. One skilled in the art will appreciate that the
appropriate dosage levels for treatment, according to certain
embodiments, will thus vary depending, in part, upon the molecule
delivered, the indication for which a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1,
with or without at least one additional therapeutic agent, is being used,
the route of administration, and the size (body weight, height, body
surface and/or organ size) and/or condition (the age, physical condition,
and/or general health) of the patient. In certain embodiments, the
clinician will consider the severity and history of the disease for which
a specific binding agent to RANKL, a specific binding agent to TNF,
and/or a specific binding agent to IL-1R1, with or without at least one
additional therapeutic agent, is being used. In certain embodiments, the
clinician may titer the dosage and modify the route of administration to
obtain the optimal therapeutic effect. In certain embodiments, a typical
dosage may range from about 0.1 μg/kg to up to about 100 mg/kg or
more, depending on the factors mentioned above. In certain embodiments, a
higher dosage of specific binding agent to RANKL, specific binding agent
to TNF, and/or specific binding agent to IL-1R1 is used with increasing
weight of the patient undergoing therapy. In certain embodiments, the
dosage may range from 0.1 μg/kg up to about 100 mg/kg; or 1 μg/kg
up to about 100 mg/kg; or 5 μg/kg up to about 100 mg/kg.

[0201]In certain embodiments, the frequency of dosing will take into
account the pharmacokinetic parameters of a specific binding agent to
RANKL, a specific binding agent to TNF, and/or a specific binding agent
to IL-1R1 and/or any additional therapeutic agents in the formulation
used. In certain embodiments, a clinician will administer the composition
until a dosage is reached that achieves the desired effect. In certain
embodiments, the composition may therefore be administered as a single
dose, or as two or more doses (which may or may not contain the same
amount of the desired molecule) over time, or as a continuous infusion
via an implantation device or catheter. Further refinement of the
appropriate dosage is routinely made by those of ordinary skill in the
art and is within the ambit of tasks routinely performed by them. In
certain embodiments, the effective dosage of a specific binding agent to
RANKL, a specific binding agent to TNF, and/or a specific binding agent
to IL-1R1 used for treatment increases over the course of a patient
treatment. In certain embodiments, the effective dosage of a specific
binding agent to RANKL, a specific binding agent to TNF, and/or a
specific binding agent to IL-1R1 used for treatment decreases over the
course of a patient treatment. In certain embodiments, appropriate
dosages may be ascertained through use of appropriate dose-response data.

[0202]In certain embodiments, the dosing regimen includes an initial
administration of a therapeutically effective dose of a specific binding
agent to RANKL, a specific binding agent to TNF, and/or a specific
binding agent to IL-1R1, with or without at least one additional
therapeutic agent, on days 1, 7, 14, and 21 of a treatment period. In
certain embodiments, the dosing regimen includes an initial
administration of a therapeutically effective dose of a specific binding
agent to RANKL, a specific binding agent to TNF, and/or a specific
binding agent to IL-1R1, with or without at least one additional
therapeutic agent, on days 1, 2, 3, 4, 5, 6, and 7 of a week in a
treatment period. In certain embodiments, the dosing regimen includes an
initial administration of a therapeutically effective dose of a specific
binding agent to RANKL, a specific binding agent to TNF, and/or a
specific binding agent to IL-1R1, with or without at least one additional
therapeutic agent, on days 1, 3, 5, and 7 of a week in a treatment
period. In certain embodiments, the dosing regimen includes an initial
administration of a therapeutically effective dose of a specific binding
agent to RANKL, a specific binding agent to TNF, and/or a specific
binding agent to IL-1R1, with or without at least one additional
therapeutic agent, on days 1 and 3 of a week in a treatment period. In
certain embodiments, the dosing regimen includes an initial
administration of a therapeutically effective dose of a specific binding
agent to RANKL, a specific binding agent to TNF, and/or a specific
binding agent to IL-1R1, with or without at least one additional
therapeutic agent, on day 1 of a week in a treatment period. In certain
embodiments, the treatment period comprises 1 week, 2 weeks, 3 weeks, one
month, 3 months, 6 months, one year, or more. In certain embodiments,
treatment periods are subsequent or separated from each other by one day,
one week, 2 weeks, one month, 3 months, 6 months, one year, or more.

[0203]In certain embodiments, the same therapeutically effective dose of a
specific binding agent to RANKL, a specific binding agent to TNF, and/or
a specific binding agent to IL-1R1 is administered at each dosing over
the course of a treatment period. In certain embodiments, different
therapeutically effective doses of a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1
are administered at each dosing over the course of a treatment period. In
certain embodiments, the same therapeutically effective dose of a
specific binding agent to RANKL, a specific binding agent to TNF, and/or
a specific binding agent to IL-1R1 is administered at certain dosings
over the course of a treatment period and different therapeutically
effective doses are administered at certain other dosings.

[0204]In certain embodiments, the initial therapeutically effective dose
of a specific binding agent to RANKL, a specific binding agent to TNF,
and/or a specific binding agent to IL-1R1 is in a lower dosing range, for
example, from 0.1 μg/kg up to 20 mg/kg, with subsequent doses in an
upper dosing range, for example, from 20 mg/kg up to 100 mg/kg. In
certain embodiments, the initial therapeutically effective dose of a
specific binding agent to RANKL, a specific binding agent to TNF, and/or
a specific binding agent to IL-1R1 is in an upper dosing range, for
example, from 20 mg/kg up to 100 mg/kg, with subsequent doses in a lower
dosing range, for example, from 0.1 μg/kg up to 20 mg/kg. Those ranges
and any ranges discussed in this application include the endpoints and
all values between the endpoints.

[0205]In certain embodiments, the initial therapeutically effective dose
of a specific binding agent to RANKL, a specific binding agent to TNF,
and/or a specific binding agent to IL-1R1 is administered as a "loading
dose." "Loading dose" refers to an initial dose of a specific binding
agent to RANKL, a specific binding agent to TNF, and/or a specific
binding agent to IL-1R1 that is administered to a patient, where the dose
of the specific binding agent to RANKL, the specific binding agent to
TNF, and/or the specific binding agent to IL-1R1 administered falls
within a higher dosing range, for example, 20 mg/kg up to 100 mg/kg. That
range and any ranges discussed in this application include the endpoints
and all values between the endpoints. In certain embodiments, the loading
dose is administered as a single administration, for example, including,
but not limited to, a single infusion administered intravenously. In
certain embodiments, the loading dose is administered as multiple
administrations, for example, including, but not limited to, multiple
infusions administered intravenously. In certain embodiments, the loading
dose is administered over a 24-hour period. In certain embodiments, after
administration of the loading dose, the patient is administered one or
more additional therapeutically effective doses of the specific binding
agent to RANKL, the specific binding agent to TNF, and/or the specific
binding agent to IL-1R1. In certain such embodiments, subsequent
therapeutically effective doses of the specific binding agent to RANKL,
the specific binding agent to TNF, and/or the specific binding agent to
IL-1R1 are administered according to a weekly dosing schedule, for
example, but not limited to, once every two weeks, once every three
weeks, or once every four weeks. In certain such embodiments, the dose of
subsequent therapeutically effective doses falls within a lower dosing
range, for example, 0.1 μg/kg up to 20 mg/kg.

[0206]In certain embodiments, after administration of the loading dose,
the patient is administered one or more additional therapeutically
effective doses of the specific binding agent to RANKL, the specific
binding agent to TNF, and/or the specific binding agent to IL-1R1
according to a "maintenance schedule." Exemplary maintenance schedules
include, but are not limited to, administration once a month, once every
six weeks, once every two months, once every ten weeks, once every three
months, once every 14 weeks, once every four months, once every 18 weeks,
once every five months, once every 22 weeks, once every six months, once
every seven months, once every eight months, once every nine months, once
every ten months, once every eleven months, or once every twelve months.
In certain embodiments, subsequent doses are administered at more
frequent intervals, for example, once every two weeks to once every
month. In certain such embodiments, subsequent doses of a specific
binding agent to RANKL, a specific binding agent to TNF, and/or a
specific binding agent to IL-1R1 fall within a lower dosing range, for
example, 0.1 μg/kg up to 20 mg/kg. In certain embodiments, subsequent
doses are administered at less frequent intervals, for example, once
every month to once every twelve months. In certain such embodiments,
subsequent doses of a specific binding agent to RANKL, a specific binding
agent to TNF, and/or a specific binding agent to IL-1R1 fall within a
higher dosing range, for example, 20 mg/kg up to 100 mg/kg.

[0207]In certain embodiments, the route of administration of the
pharmaceutical composition is in accord with known methods, e.g. orally,
through injection by intravenous, intraperitoneal, intracerebral
(intra-parenchymal), intracerebroventricular, intramuscular,
intra-ocular, intraarterial, intraportal, or intralesional routes; by
sustained release systems or by implantation devices. In certain
embodiments, the compositions may be administered by bolus injection or
continuously by infusion, or by implantation device.

[0208]In certain embodiments, intravenous administration occurs by
infusion over a period of 1 to 10 hours. In certain embodiments,
intravenous administration occurs by infusion over a period of 1 to 8
hours. In certain embodiments, intravenous administration occurs by
infusion over a period of 2 to 7 hours. In certain embodiments,
intravenous administration occurs by infusion over a period of 4 to 6
hours. Those ranges and any ranges discussed in this application include
the endpoints and all values between the endpoints. In certain
embodiments, the infusion period depends on the specific binding agent to
RANKL, the specific binding agent to TNF, and/or the specific binding
agent to IL-1R1 to be administered. The determination of certain
appropriate infusion periods is within the skill of the art. In certain
embodiments, the initial infusion is given over a period of 4 to 6 hours,
with subsequent infusions delivered more quickly. In certain such
embodiments, subsequent infusions are administered over a period of 1 to
6 hours.

[0209]In certain embodiments, a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1
and/or any additional therapeutic agents can be placed into syringes and
stoppered such that the prefilled syringes have a minimized headspace. In
certain embodiments, the specific binding agent to RANKL is an antibody
which specifically binds RANKL. In certain embodiments, the antibody is
αRANKL-1. In certain embodiments, the specific binding agent to TNF
is a soluble TNF receptor. In certain embodiments, the soluble TNF
receptor is sTNFR:Fc. In certain embodiments, the specific binding agent
to IL-1R1 is an antibody which specifically binds IL-1R1. In certain
embodiments, the antibody is selected from 15C4, 26F5 and 27F2 as
described in U.S. Publication No. 2004/0097712. In certain embodiments,
syringes containing a specific binding agent to RANKL, a specific binding
agent to TNF, and/or a specific binding agent to IL-1R1 are stoppered
with Fluorotec/B2 coated plungers, for example, including, but not
limited to Daikyo/West (Becton Dickinson, part numbers 47165910 and
47165919) and Dupont (Becton Dickinson, part numbers 5080958 and 5115079)
using either a vacuum stopper placement method or a mechanical stopper
placement method, as described below.

[0210]In certain embodiments, a vacuum stopper placement method includes
use of a vacuum stopper placement unit, for example, including, but not
limited to Autoclavable Stopper Placement Unit (ASPU), ImproSystems Hypak
filler, catalog number 897400. In certain embodiments, syringes
containing a specific binding agent to RANKL, a specific binding agent to
TNF, and/or a specific binding agent to IL-1R1 are placed in the unit and
stoppered under 75 pounds per square inch inlet pressure with vacuum
cycle settings of FC1--21'' Hg, FC2--6.5'' Hg, FC3 26.5'' Hg. In certain
embodiments, those settings result in at least a 3 mm headspace. In
certain embodiments, stoppered and prefilled syringes with minimized
headspace are produced from stoppered and prefilled syringes having at
least a 3 mm headspace by manually manipulating such stoppered and
prefilled syringes to express air from the needle by orienting the
syringe with the needle up such that the bubble rises to the base of the
needle, expelling the air out of the needle, and reshielding of the
needle.

[0211]In certain embodiments, a mechanical stopper placement method
includes use of a mechanical stopper placement unit, for example,
including, but not limited to, Groninger, model SVH200. In certain
embodiments, syringes containing a specific binding agent to RANKL, a
specific binding agent to TNF, and/or a specific binding agent to IL-1R1
are placed in the unit and stoppers are mechanically positioned. In
certain embodiments, stoppers are positioned using a vacuum. In certain
embodiments, a vent tube is used during the stoppering process. To
produce stoppered and prefilled syringes with minimized headspace, the
stoppers are positioned against the upper surface of the liquid
composition containing a specific binding agent to RANKL, a specific
binding agent to TNF, and/or a specific binding agent to IL-1R1 such that
the stopper is as close as possible to the liquid surface with a maximum
of contact between the bottom surface of the stopper and the upper
surface of the liquid. In certain embodiments, the distance between the
bottom surface of the stopper and the meniscus is minimized.

[0212]In certain embodiments, the headspace of a prefilled and stoppered
syringe is measured manually with a calibrated caliper. An exemplary
method of calibrating a caliper is to place it in a fully closed position
(0.00'') and then calibrate with gauge blocks 0.050'' and 4.000''
according to the manufacturer's instructions. In certain embodiments, the
headspace of a prefilled and stoppered syringe is measured with a
microscope and microscope ruler. In certain such embodiments, calipers
are used to record the distance between the top of the meniscus to the
bottom of the flat body of the plunger using calipers. In certain
embodiments, the headspace of a prefilled and stoppered syringe is
measured with an optical comparator. An exemplary optical comparator is
Deltronic DH 216, Horizontal Optical Comparator. In certain such
embodiments, measurements are made by placing the syringe in a vertical
position and parallel to the optical lens. A magnified image is projected
onto a screen for inspection. Calipers on the optical comparator are used
to record the distance between the top of the meniscus to the bottom of
the flat body of the plunger. In certain embodiments, the headspace is
the distance in millimeters from the top of the meniscus to the bottom of
the flat body of the plunger.

[0213]In certain prefilled syringes, the headspace varies from 2 mm to 5
mm. In certain prefilled syringes, the headspace is 3 mm±0.00254 mm.
In certain prefilled syringes having a minimized headspace, the headspace
is less then 2.9 mm, or less than 2.7 mm, or less than 2.5 mm, or less
than 2.3 mm, or less than 2 mm, or less than 1.5 mm, or less than 1.0 mm,
or there is no detectable headspace.

[0214]In certain embodiments, syringe barrels comprise material such as,
but not limited to, glass, cyclic olefin polymer ("COP"), or cyclic
olefin copolymer ("COC"). In certain embodiments, a silicone coating is
applied to a syringe barrel. In certain such embodiments, the silicone
coating is cross-linked silicone, baked high viscosity silicone, or
sprayed-on silicone oil. In certain embodiments, the silicone coating is
applied by the syringe manufacturer of the syringe. Certain syringe
manufacturers include, but are not limited to, Daikyo, Schott-Form a
Vitrum, Bunder, and Becton-Dickinson. In certain embodiments, syringe
barrels do not comprise a silicone coating.

[0215]In certain embodiments, a syringe plunger is coated. Exemplary
syringe plunger coatings include, but are not limited to,
polytetrafluoroethylene (PTFE), Teflon®, and ethylene
tetrafluoroethylene (ETFE), Fluorotec®. In certain embodiments, the
coating is applied by the manufacturer. Certain manufacturers include,
but are not limited to, Daikyo and Becton-Dickinson.

EXAMPLES

Example 1

[0216]The following experiments were performed to evaluate the stability
of specific binding agent compositions stored in containers under certain
conditions. Stability was monitored under static storage conditions and
after shipping. Specifically, certain aspects of syringes were
investigated to identify parameters that affect protein aggregation,
which, under certain conditions, lead to visible particle formation in
the compositions. Various silicone coatings of containers and closures of
prefilled syringes were investigated. The specific binding agent used in
the experiments below was αRANKL-1.

General Methods

[0217]The concentration of αRANKL-1 in the following experiments
varied between 30 mg/ml and 105 mg/ml. αRANKL-1 was formulated in
10 mM sodium acetate, 5% sorbitol, pH 5.2. For experiments using vials,
compositions were placed into 3-cc vials to a final volume of 1 ml. For
experiments using syringes, 1 ml syringes were used. Compositions in
containers were stored for up to 24 months. Compositions in containers
were monitored for antibody monomer, high molecular species (aggregates),
or low molecular weight species (for example, molecules created by
clipping) by native SEC-HPLC or non-reduced, denaturing SEC-HPLC. Visible
particles were assessed in compositions in containers by visual
inspection of containers as described below.

[0218]Native SEC-HPLC was performed using two TSKgel G3000-SWxL 7.8
mm×300 mm columns (Tosoh Bioscience) employed in tandem, with 5
μm particle size and pore size of 250 Å, on an Agilent 1100 Series
HPLC with diode array detection. The mobile phase was 100 mM sodium
phosphate, 500 mM sodium chloride, 5% ethanol, pH 7.0. The flow rate was
0.5 ml/minute. The sample load was 120 μg protein, and the column
eluate was monitored at 235 nm and at 280 nm. Integrated peak areas in
the chromatograms were used to quantify the amounts of monomer, which
elutes with the main peak; and high molecular weight species, also
referred to as aggregates, which elutes with the pre-peak.

[0219]Visual inspection of containers for visible particles was conducted
in a visual inspection cabinet, the Phoenix Imaging Manual Inspection
Booth, catalog no. MIB-100. The visual inspection cabinet has separate,
non-reflective black and white surfaces. The black and white surfaces are
of sufficient size to serve as a background for the entire container
during the inspection process. The visual inspection cabinet also has a
light source that provides illumination of at least 2000 Lux at the
position of the sample.

[0220]To inspect for visible particles, containers were gently swirled or
inverted while holding the sample upright at eye level in the visual
inspection cabinet. Care was taken to ensure that air bubbles were not
introduced while swirling or inverting the containers. Each container was
visually observed for approximately five seconds in front of the white
surface. Then each container was visually observed for approximately five
seconds in front of the black surface. In some cases, a magnifying glass
was used, in addition to the light source, to confirm the presence or
absence of visible particles.

[0221]The presence or absence of visible particles was observed as
described above. Then a particle score was assigned to each container and
recorded as follows. A score of 0 indicates no particles observed; a
score of 1 indicates one or two particles observed; a score of 2
indicates three to nine particles observed; a score of 3 indicates ten to
49 particles observed; a score of 4 indicates 50 or more particles
observed.

Stability in Glass Vials Under Static Storage Conditions

[0222]FIG. 1 shows the results of native SEC-HPLC analysis of
αRANKL-1 compositions at a protein concentration of either 70 mg/ml
or 105 mg/ml, stored in glass vials for 24 months under static
conditions, and analyzed at various time points as indicated in the
figure. Three different lots were analyzed (lots A, B, and C). FIG. 1 (A)
shows the % main peak (monomer) and FIG. 1 (B) shows the aggregate
(pre-peak). The results indicate that αRANKL-1 shows little
aggregate formation when stored in glass vials at 4° C. for up to
24 months under static conditions. The figure also shows that similar
results were obtained for formulations containing 70 mg/ml protein and
for formulations containing 105 mg/ml protein.

Stability in Prefilled Glass Syringes Under Static Storage Conditions

[0223]FIG. 2 shows the results of native SEC-HPLC of αRANKL-1
compositions at different protein concentrations, stored in either
prefilled glass luer lock syringes or prefilled glass staked-needle
syringes, and analyzed at various time points as indicated in the figure.
The results indicate that αRANKL-1 shows little aggregate formation
when stored under static conditions in either prefilled glass luer lock
syringes or prefilled glass staked-needle syringes at 4° C. for up
to 24 weeks. The figure also shows that similar results were obtained for
formulations containing 30 mg/ml protein, 70 mg/ml protein, and 105 mg/ml
protein.

Stability in Prefilled Glass Syringes after Shipping

[0224]In contrast to the stability results discussed above, prefilled
glass staked needle syringes containing αRANKL-1 at 60 mg/ml
protein and shipped by air at a temperature between 2° C. and
8° C., for a distance of 1050 miles, showed visible particles
after shipping, as assessed by visual inspection (data not shown).

Effect of Silicone Coating of Containers and Closures on Shipping
Stability

[0225]To investigate the effects of various syringe and plunger materials
and coatings on particle formation in prefilled syringes after shipping,
the following experiment was performed. An αRANKL-1 composition at
60 mg/ml protein was placed into different types of containers having
different types of closures, each having different silicone and other
coatings as indicated in Tables 1 and 2. The manufacturers and the
catalog numbers for the containers and closures used in these experiments
are provided in Tables 1 and 2. Containers comprised of glass, cyclic
olefin polymer ("COP;" [Resin Cz®]), or cyclic olefin copolymer
("COC") were tested. Three different silicone coatings were tested:
baked-on high viscosity silicone, cross-linked silicone, and sprayed-on
silicone oil. Certain containers did not comprise a silicone coating. Two
different closure coatings were tested: polytetrafluoroethylene (PTFE),
Teflon® and ethylene tetrafluoroethylene (ETFE), Fluorotec®.

[0226]Each experimental group listed in Table 3 consisted of 10
containers. The containers were stored at 4° C. for up to one week
before being subjected to shipping conditions. The shipping conditions
were by air at a temperature ranging from 2° C. to 8° C.
within C167 polyurethane shippers according to conditions specified by
the American Society for Testing and Materials (ATSM). Prefilled syringes
were shipped by air from Thousand Oaks, Calif. to Boulder, Colo., then
from Boulder, Colo. to Thousand Oaks, Calif., for a total of two airplane
flights (two air pressure cycles; each flight having one air pressure
cycle, for take-off and for landing). After shipping, visible particles
were assessed in each of the containers by visual inspection. After
inspection, a particle score was assigned to each container as described
above under General Methods. The results (for all 10 containers in each
group) are shown in Table 3.

[0227]The results indicate that the particle score was 0 or 1 in the COP
syringes, each of which comprised a barrel made of a high molecular
weight plastic material lacking silicone. Table 3, group 1. The group 1
syringe closures were coated with PTFE, which lacks silicone. Table 3,
group 1. In addition, the particle score was 0 or 1 in COC syringes, each
of which comprised a barrel coated with cross-linked silicone. Table 3,
group 2. The group 2 syringe closures were coated with Fluorotec B2,
which lacks silicone. The particle score was also 0 or 1 in glass
syringes, each of which comprised barrels either lacking silicone, or
coated with baked-on high viscosity silicone, and having closures coated
with either Fluorotec B2 lacking silicone, or Fluorotec B2 and
cross-linked silicone. Table 3, groups 3 and 5. In addition, the particle
score was 0 or 1 in glass vials lacking silicone, and having closures
coated with Fluorotec B2 and cross-linked silicone. Table 3, group 6. In
contrast, glass syringes comprising a barrel coated with sprayed-on
silicone oil and having closures coated with Fluorotec B2 and
cross-linked silicone had a particle score of 4, corresponding to the
greatest amount of visible particles. Table 3, group 4. Thus, those
results suggest that silicone from prefilled syringe barrels coated with
sprayed-on silicone oil contributes to formation of visible particles
during shipping.

[0228]In addition, two different methods of sterilization were carried out
according to standard procedures that are known in the art. Those methods
were: E-beam (gamma irradiation) and steam. Two different levels of
E-beam sterilization were tested, 15 kGy and 25 kGy. It was found that
the neither the sterilization method nor the level of E-beam
sterilization affected the particle score (data not shown).

[0229]In the following experiments, the concentration of αRANKL-1
was either 60 mg/ml or 120 mg/ml. αRANKL-1 was formulated in 10 mM
sodium acetate, 5% sorbitol, pH 5.2. αRANKL-1 compositions were
sterile filtered by passing the solution through a 0.2 μM cellulose
filter. Samples (1.0 ml) were then manually added into 1 ml COP (Resin
CZ®) plastic syringes (see Table 1). Syringes with samples in them
were stoppered with Fluorotec coated plungers (see Table 2) according to
a vacuum stopper placement method as described below.

[0231]In addition, two different methods of sterilization were carried out
according to standard procedures that are known in the art. Those methods
were: electronic beam (E-Beam) at two different energy levels, 15 kGy or
25 kGy, and steam.

[0232]Following aseptically placing of samples in them, and the stoppering
procedure, prefilled syringes were stored under static conditions or were
subjected to shipping conditions followed by storage under static
conditions. The static storage conditions were storage at 4° C.
for up to 52 weeks. The shipping conditions were by air at a temperature
ranging from 2° C. to 8° C. within C167 polyurethane
shippers according to conditions specified by the American Society for
Testing and Materials (ATSM). Prefilled syringes were shipped by air from
Thousand Oaks, Calif. to Memphis, Tenn., then from Memphis, Tenn. to
Puerto Rico, then from Puerto Rico to Memphis, Tenn., and finally from
Memphis, Tenn. to Thousand Oaks, Calif., for a total of four airplane
flights (four air pressure cycles; each flight having one air pressure
cycle, for take-off and for landing). After shipping, the prefilled
syringes were stored under static storage conditions at 4° C. for
up to 52 weeks.

[0233]At each timepoint as indicated in FIG. 3, samples were removed from
each prefilled syringe for monitoring of antibody monomer, high molecular
weight species (aggregates), or low molecular weight species (for
example, dimer molecules) by native SEC-HPLC. Native SEC-HPLC was
performed using two TSKgel G3000-SWxL 7.8 mm×300 mm columns (Tosoh
Bioscience) employed in tandem, with 5 μm particle size and pore size
of 250 Å, on an Agilent 1100 Series HPLC with diode array detection.
The mobile phase was 100 mM sodium phosphate, 500 mM sodium chloride, 5%
ethanol, pH 7.0. The flow rate was 0.5 ml/minute. The sample load was 120
μg protein, and the column eluate was monitored at 235 nm and at 280
nm. Integrated peak areas in the chromatograms were used to quantify the
amounts of monomer, which elutes with the main peak; and high molecular
weight species, also referred to as aggregates, which elutes with the
pre-peak.

[0234]FIG. 3 shows the results of the experiments as analyzed by native
SEC-HPLC. In FIG. 3, the % main peak (monomer) is shown at each timepoint
for each condition tested. The results indicate that αRANKL-1
showed little aggregate formation when placed into COP (Resin CZ®)
plastic syringes, stoppered according to a vacuum stopper placement
method to form a >3 mm headspace, and stored either under static
conditions or subjected to shipping conditions. In addition, two
different methods of sterilization were carried out according to standard
procedures that are known in the art. Those methods were: E-beam (gamma
irradiation) and steam. Two different levels of E-beam sterilization were
tested, 15 kGy and 25 kGy. The method of sterilization used in these
experiments also did not affect the results.

Example 2

[0235]The results discussed above in Example 1 suggested that plunger
movement during shipping of certain prefilled containers contributes to
protein aggregation, which may lead to formation of visible particles in
the composition. Therefore, parameters that contribute to plunger
movement during shipping were considered. One such parameter is
headspace. It was hypothesized that the smaller the headspace, the less
the amount of plunger movement and consequently, according to the
hypothesis, less visible particles would be observed in the composition
after shipping. To test that hypothesis, the following experiment was
carried out.

[0236]The following experiments were performed to assess the effects of
minimized headspace on formation of visible particles during shipping of
prefilled syringes containing specific binding agent compositions.
Different methods of placing compositions in syringes and stoppering
syringes to produce minimized headspace were investigated. The specific
binding agents used in the experiments below were either sTNFR:Fc or
αRANKL-1.

[0240]To manually produce stoppered and prefilled syringes with minimized
headspace, the stoppered and prefilled syringes from the unit were
manually manipulated to express air from the needle by orienting the
syringe with the needle up such that the bubble rises to the base of the
needle, expelling the air out of the needle, and reshielding of the
needle. As a control for that procedure, a control group of stoppered and
prefilled syringes were manually manipulated to express air from the
needle, then the plunger was pulled back to approximate the original
stopper position and form a >3 mm headspace followed by reshielding of
the needle.

[0241]For the mechanical stopper placement method, a mechanical stopper
placement unit (Groninger, model SVH200) was used. Syringes containing
samples were placed in the unit and stoppers were mechanically
positioned. To perform this method, a stopper placement tube of smaller
diameter than the syringe placed the stopper within the syringe barrel.
The stopper placement tube was then retracted, and the stopper expanded
to fill the syringe barrel. To produce stoppered and prefilled syringes
with minimized headspace, the stoppers were positioned against the upper
surface of the liquid composition such that the stopper was as close as
possible to the liquid surface with a maximum of contact between the
bottom surface of the stopper and the upper surface of the liquid.

[0242]The headspace for each prefilled and stoppered syringe was measured
manually with a calibrated caliper. The caliper was calibrated by placing
it in a fully closed position (0.00'') and then calibrating with gauge
blocks 0.050'' and 4.000''according to the manufacturer's instructions.
The headspace is the distance in millimeters from the top of the meniscus
to the bottom of the flat body of the plunger. In certain prefilled
syringes, the headspace varied from 2 mm to 5 mm. In certain prefilled
syringes, the headspace was 3 mm±0.001 0.00254 mm. In certain
prefilled syringes having a minimized headspace, the headspace was less
than 2 mm. In certain prefilled syringes having a minimized headspace,
the headspace was less than 1.3 mm.

[0243]Prefilled syringes were packaged in boxes and shipped by air at a
temperature ranging from 2° C. to 8° C. within C167
polyurethane shippers according to conditions specified by the American
Society for Testing and Materials (ATSM). Prefilled syringes were shipped
by air from Thousand Oaks, Calif. to Memphis, Tenn., then from Memphis,
Tenn. to Puerto Rico, then from Puerto Rico to Memphis, Tenn., and
finally from Memphis, Tenn. to Thousand Oaks, Calif., for a total of four
airplane flights (four air pressure cycles; each flight having one air
pressure cycle, for take-off and for landing). The total transit time was
four days or less.

[0244]Visual inspection of containers for visible particles was conducted
in a visual inspection cabinet the Phoenix Imaging Manual Inspection
Booth, catalog no. MIB-100. The visual inspection cabinet has two
separate surfaces that are each used as a background for visual
inspection of a container. One surface is a non-reflective white surface
and the second surface is a non-reflective black surface. The white and
black surfaces are of sufficient size so that they may be used as a
background for the entire container during the inspection process. The
visual inspection cabinet has a light source that provides illumination
of at least 2000 Lux at the position of the sample.

[0245]To inspect for visible particles, containers were gently swirled or
inverted while holding the sample upright at eye level in the visual
inspection cabinet. Care was taken to ensure that air bubbles were not
introduced while swirling or inverting the containers. Each container was
visually observed for approximately five seconds in front of the white
surface. Then each container was visually observed for approximately five
seconds in front of the black surface. In some cases, a magnifying glass
was used, in addition to the light source, to confirm the presence or
absence of visible particles.

[0246]The presence or absence of visible particles was observed as
described above. Then a particle score was assigned to each container and
recorded as follows. A score of 0 indicates no particles observed; a
score of 1 indicates one or two particles observed; a score of 2
indicates three to nine particles observed; a score of 3 indicates ten to
49 particles observed; a score of 4 indicates 50 or more particles
observed.

[0247]The results of experiments with prefilled syringes containing
αRANKL-1 compositions are shown in Table 4 below. The results show
that none of the syringes containing an αRANKL-1 composition and
stoppered according to the vacuum stopper placement method to form either
a >3 mm headspace or a minimized headspace had visible particles after
shipping. The results also show that none of the syringes containing an
αRANKL-1 composition and stoppered according to the mechanical
stopper placement method to form a minimized headspace had visible
particles after shipping.

[0248]The results of experiments with prefilled syringes containing
sTNFR:Fc are shown in Table 5. The results show that all syringes
containing a sTNFR:Fc composition and stoppered according to the vacuum
stopper placement method to form a >3 mm headspace had visible
particles after shipping. 29 (of 30 total) prefilled syringes had a
particle score of 3 and one prefilled syringe had a particle score of 2.
The results also show that syringes stoppered according to the vacuum
stopper placement method to form a minimized headspace reduced the number
of visible particles observed after shipping. In that experiment, 29
prefilled syringes had a particle score of 0, while two prefilled
syringes had a particle score of 2. The two prefilled syringes that had a
particle score of 2 also had a small air bubble remaining after the air
was expressed suggesting that the headspace for those syringes was not
minimized. In addition, ten prefilled control syringes were tested. The
prefilled control syringes were first stoppered according to the vacuum
stopper placement method to form a minimized headspace. That method was
then followed by repositioning of the plunger to form a >3 mm
headspace. As shown in Table 5, all ten prefilled control syringes had
visible particles after shipping and each had a particle score of 3.

[0249]The results in Table 5 also show that all syringes containing a
sTNFR:Fc composition and stoppered according to the mechanical stopper
placement method to form a >3 mm headspace had visible particles after
shipping. Six (of 10 total) prefilled syringes had a particle score of 3
and four prefilled syringes had a particle score of 2. In addition, the
results in Table 5 show that syringes stoppered according to the
mechanical stopper placement method to form a minimized headspace reduced
the number of visible particles observed after shipping. In that
experiment, all 30 prefilled syringes had a particle score of 0.

[0250]In summary, the results of stoppering syringes according to two
different methods to form a minimized headspace suggested that
manufacturing syringes containing specific binding agent compositions and
stoppering them according to a method to form a minimized headspace is
desirable to reduce or eliminate formation of visible particles during
shipping.

[0255]For the mechanical stopper placement method, a mechanical stopper
placement unit (Groninger, model SVH200) was used. Syringes containing
samples were placed in the unit and stoppers were mechanically
positioned. To perform this method, a stopper placement tube of smaller
diameter than the syringe placed the stopper within the syringe barrel.
The stopper placement tube was then retracted, and the stopper expanded
to fill the syringe barrel. To produce stoppered and prefilled syringes
with minimized headspace, the stoppers were positioned against the upper
surface of the liquid composition such that the stopper was as close as
possible to the liquid surface with a maximum of contact between the
bottom surface of the stopper and the upper surface of the liquid.

[0256]The headspace for each prefilled and stoppered syringe was measured
manually with a calibrated caliper as described in the section entitled
"Visible Particle Analysis" above.

[0257]Three prefilled syringes were tested, each under different
conditions. A sTNFR:Fc composition was added to one syringe and the
syringe was stoppered according to the vacuum stopper placement method to
form a >3 mm headspace and was stored under static conditions (FIG. 4,
green line, designated "unshipped control"). A sTNFR:Fc composition was
also added to a second syringe and the syringe was stoppered according to
the vacuum stopper placement method to form a >3 mm headspace and was
subjected to shipping conditions as described below (FIG. 4, blue line,
designated "shipped control"). A sTNFR:Fc composition was added to the
third syringe and the syringe was stoppered according to the mechanical
stopper placement method, followed by the procedure described above under
the subtitle "visible particle analysis," to form a minimized headspace
(FIG. 4, red line, designated "shipped, minimized headspace"), and was
subjected to shipping conditions as follows. For shipping, the prefilled
syringes were packaged in boxes and shipped by air at a temperature
ranging from 2° C. to 8° C. within C167 polyurethane
shippers according to conditions specified by the American Society for
Testing and Materials (ATSM). Prefilled syringes were shipped by air from
Thousand Oaks, Calif. to Memphis, Tenn., then from Memphis, Tenn. to
Puerto Rico, then from Puerto Rico to Memphis, Tenn., and finally from
Memphis, Tenn. to Thousand Oaks, Calif., for a total of four airplane
flights (four air pressure cycles; each flight having one air pressure
cycle, for take-off and for landing). The total transit time was four
days or less.

[0258]To measure sub-visible particle size using the Malvern Zetasizer
instrument, 1 ml sample volumes were placed in a disposable cuvette and
measurements were performed at 25° C. Each 1 ml sample was
analyzed by five sub-runs of 10 seconds each. A sub-run is a replicate
measurement of each sample. Hydrodynamic diameter and polydispersity
values were calculated using Dispersants Manager software, using a
dispersant viscosity of 0.939 cP.

[0259]The intensity weighted size distribution is shown in FIG. 4.
Intensity-weighted size distribution is the signal based on the intensity
of the light scattered. The results show that the sample from the
prefilled syringe designated "shipped control" in FIG. 4 (blue line), had
a bimodal distribution with a distinct new peak of large hydrodynamic
size. The results also show that the sample from the prefilled syringe
designated "unshipped control" in FIG. 4 (green line), and the sample
from the prefilled syringe designated "shipped, minimized headspace" in
FIG. 4 (red line), did not have the distinct new peak of large
hydrodynamic size.

[0260]The numerical results of the same experiment are presented in Table
6. Those results show that the sample from the prefilled syringe
designated "shipped control" had a larger z-average hydrodynamic diameter
and greater polydispersity compared to the sample from the prefilled
syringe designated "unshipped control" and the prefilled syringe
designated "shipped, minimized headspace".

[0261]The results of these experiments suggested that sub-visible
particles were not present in the prefilled syringe after shipping, when
the syringe was stoppered according to the mechanical stopper placement
method to form a minimized headspace. The results thus suggested that
manufacturing syringes containing specific binding agent compositions and
stoppering them according to a method to form a minimized headspace is
desirable to reduce or eliminate formation of particles, including
visible and sub-visible particles, during shipping.